Laboratory Manual For Genetic Engineering

Laboratory Manual
Genetic
for
Engineering
S. John Vennison
Laboratory Manual
for
Genetic Engineering
S. John Vennison
Lecturer
Department of Biotechnology
Anna University
Tiruchirappalli
New Delhi-110001
2009
LABORATORY MANUAL FOR GENETIC ENGINEERING
S. John Vennison
© 2009 by PHI Learning Private Limited, New Delhi. All rights reserved. No part of this book may
be reproduced in any form, by mimeograph or any other means, without permission in writing from
the publisher.
ISBN-978-81-203-3814-2
The export rights of this book are vested solely with the publisher.
Published by Asoke K. Ghosh, PHI Learning Private Limited, M-97, Connaught Circus,
New Delhi-110001 and Printed by Glorious Printer, Delhi-110092.
Contents
Preface .......................................................................................................................................................................... vii

1. Isolation of Genomic DNA from GRAM Negative, GRAM Positive
Bacteria, Blood and Mammalian Tissue ....................................................................... 1-6
1.1 Total DNA Extraction from Agrobacterium Tumefaciens and E. coli 1
1.2 Total Cell DNA Isolation from Bacillus Thuringiensis 2
1.3 Genomic DNA Isolation from Blood 4
1.4 A Rapid Procedure for Isolation of DNA Cultured Mammalian Cells 5
References 6
2. Isolation of Plasmid DNA from GRAM Negative and
GRAM Positive Bacteria ............................................................................................... 7-21
2.1 Growth of Bacteria 7
2.2 Harvesting and Lysing of Bacteria 8
2.3 Purification of the Plasmid DNA 8
2.3.1 Cloning in Plasmid Vectors 9
2.3.2 Cloning DNA Fragments with Protruding Ends 9
2.4 Modified Alkaline Lysis Protocol for Both E. coli and Bacillus sp. 9
2.5 High Molecular Weight Plasmid Preparation from GRAM Negative Bacteria
(Including Agrobacterium Tumefaciens) 11
2.6 Plasmid Isolation from E. coli (Mini Preparation) 12
2.7 Large Scale Plasmid Preparation for E. coli (Alkaline Lysis) 13
2.7.1 Plasmid Amplification 14
2.7.2 Harvesting 14
2.7.3 Lysis with Alkali 14
2.8 Rapid Boiling Method of Isolation of Bacterial Plasmid 14
2.9 Isolation of Plasmid DNA from Bacillus Thuringiensis 15
2.10 Purification of High Molecular Weight Plasmids from Bacillus Thuringiensis 16
2.11 Isolation of Plasmid DNA from Bacillus Subtilis and Bacillus Megaterium 17
2.12 Purification of Genomic and Plasmid DNA through Phenol
Chloroform Treatment 17
iii
iv Contents
2.13 Ultra Purification of Plasmid DNA through Cesium Chloride

Ethidium Bromide Gradient 18
References 20
3. Isolation of RNA from Bacteria and Cultured Mammalian Cells ........................ 22-25
3.1 Isolation of Bacterial RNA 23
3.2 Isolation of mRNA from Cultured Mammalian Cells 24
References 25
4. Estimation of Nucleic Acids ........................................................................................ 26-31
4.1 Estimation of DNA 26
4.1.1 UV Quantitation of DNA by UV Absorbance Spectrophotometry 26
4.1.2 TD-20/20 Luminometer Method for DNA Quantitation 27
4.1.3 Diphenylamine Method 29
4.2 Estimation of RNA 30
4.2.1 Orcinol Method 30
References 31
5. Restriction Digestion and Ligation of DNA ............................................................. 32-35
5.1 Restriction Digestion of DNA 33
5.2 Purification of Restricted DNA Fragments 33
5.3 DNA Ligation 34
References 35
6. Polymerase Chain Reaction and Randomly Amplified Polymorphic DNA ......... 36-40
6.1 Important Parameters in the PCR 36
6.1.1 Tm of Primers 37
6.1.2 Mg Concentration 37
6.1.3 Length of Expected Product 37
6.2 Polymerase Chain Reaction (PCR) 38
6.3 Random Amplified Polymorphic DNA (RAPD) 39
References 40
7. Electrophoresis of Nucleic Acids ................................................................................ 41-49
7.1 Agarose Gel Electrophoresis of DNA 41
7.1.1 The Rate of Migration of DNA through Agarose Gels 42
7.2 Polyacrylamide Gel Electrophoresis of DNA 46
7.3 Electrophoresis of RNA through Gels Containing Formaldehyde 48
References 49
8. Slot Lysis Agarose Gel Electrophoresis ..................................................................... 50-54
8.1 Horizontal Slot Lysis Electrophoresis for E. coli 50
8.2 Vertical Slot Lysis Electrophoresis for B. Thuringiensis
(Modified Eckhardt's Lysate Electrophoresis) 51
References 54
Contents v
9. Purification of DNA from Agarose and Polyacrylamide Gels ............................... 55-56

9.1 Isolation of DNA from Agarose Gels 55
9.2 Isolation of DNA Fragments from Polyacrylamide Gels 56
Reference 56
10. Transformation of GRAM Negative and GRAM Positive Bacteria
with plasmid DNA........................................................................................................ 57-68
10.1 Competent Cell Transformation of Gram Negative Bacteria 57
10.2 E. coli Transformation by Calcium Chloride Method 58
10.3 E. coli Transformation by TSB Buffer Method 59
10.4 E. coli Transformation by Electroporation 60
10.5 Simple Method of Plasmid Transformation of E. coli by Rapid Freezing 61
10.6 Protoplast Transformation of Bacillus sp. with Plasmid DNA 61
10.7 Protoplast Transformation of Bacillus sphaericus with Plasmid DNA 62
10.8 Competent Cell Transformation of Bacillus subtilis with Plasmid DNA 64
10.9 Transformation of Bacillus Thuringiensis by Electroporation 66
References 67
11. Estimation of Proteins ................................................................................................. 69-71
11.1 Estimation of Protein by Bradford's Method 69
11.2 Estimation of Protein by Lowry's Method 70
References 71
12. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Proteins ...... 72-78
12.1 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis of Proteins 73
12.2 Silver Staining of Protein Gels 77
References 78
13. -Galactosidase Assay ................................................................................................. 79-81
13.1 Disruption of Selective Permeability 80
13.2 Enzyme Assay 81
Reference 81
14. Transduction of Plasmid DNA using CP-51 and CP-54 Bacteriophages ............. 82-85
14.1 Transduction of Plasmid in Bacillus sp. with CP-51 and CP-54 Phage 82
References 85
15. Bacterial Conjugation .................................................................................................. 86-89
15.1 Conjugal Transfer of DNA into Cyanobacteria 86
15.2 Conjugal Plasmid Transfer in B. Thuringiensis 87
15.3 Introduction of Binary Plasmids into Agrobacterium by Triparental Mating 88
References 89
16. Blotting Techniques ................................................................................................................ 90-99
16.1 Western Blotting 90
16.2 Immunoblotting Assay 93
vi Contents
16.3 Southern Blotting 94

16.3.1 Capillary Blotting or Passive Diffusion Blotting on Nitrocellulose 94
16.3.2 Southern Blotting using Semiphor Blotting Unit 96
16.3.3 Colony Blotting 98
16.4 Northern Blotting 99
References 99
32
17. P Labelled Probe Preparation and Measurement of Radioactivity in
Radio-Labelled Nucleic Acid .................................................................................. 100-102
17.1 32P Labelled Probe Preparation 100
17.1.1 Nick Translation or Oligolabelling with a32P dCTP 100
17.1.2 Random Primer Labelling of Probe using a32P dCTP 101
17.1.3 Separation of Probe from Unincorporated Label by Gel-
Filtration through Sephadex G-50 Column 101
17.2 Measurement of Radioactivity in Nucleic Acid 101
17.2.1 Absorption to DE-81 Filter 101
17.2.2 Precipitation with Trichloroacetic Acid (TCA) 102
Reference 102
18. Hybridization Techniques.............................................................................................103-106
18.1 Hybridization of Southern Filters 103
18.1.1 Prehybridization 103
18.2 Colony Hybridization 105
References 106
Appendices ........................................................................................................................ 107-124
1. Stock Solutions and Working Concentrations of Antibiotics 109
2. Conversion of rpm to g 110
3. Conversion of g to rpm 111
4. Stock Solutions 112
5. DNA/Protein Conversions 117
6. Common Conversions of Oligonucleotides 118
7. Restriction Enzymes and their Cleavage Sites 120
8. Estimation of Ends (3' or 5') Concentration 122
9. Recommended Gel Percentages for Separation of Linear DNA 123
10. Calculating Primer Quantity 124
Preface
This laboratory manual is the outcome of hands-on experience in research and teaching in the
area of genetic engineering for the past eighteen years. This book consists of 18 chapters on basic
genetic engineering laboratory techniques starting from DNA isolation to DNA transformation.
I hope this laboratory manual will be useful to B. Tech and M. Tech students of biotechnology
as well as B.Sc. and M.Sc. students of biotechnology of most universities to carry out
experiments in genetic engineering and recombinant DNA technology as per prescribed
laboratory courses.
I am grateful to Prof. V. Sekar, Former Professor and Head, Department of Molecular
Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, India, for his
scholarly guidance and encouragement. I am deeply indebted to Dr. V. Ramachandran, Vice-
Chancellor, Anna University Tiruchirappalli, for his wise counsel. I thank Dr. P. Rajaguru, Head
of the Department of Biotechnology, Anna University Tiruchirappalli, for his all time encourage-
ment. I am also thankful to the research scholars Mr. P. Thirumalai Vasan, Mr. D. Immanuel Gilwax
Prabhu, Mr. S. Gowri Sankar and Mr. S. Venkatesh for their help in the preparation of the
manuscript. I express my sincere thanks to the publishers, PHI Learning Private Limited,
New Delhi and the editorial and production teams for their whole-hearted approach to bring out
this laboratory manual in time.
S. JOHN VENNISON
vii
1
Isolation of Genomic DNA from GRAM
Negative, GRAM Positive Bacteria, Blood
and Mammalian Tissue
A number of methods and technologies are available for the isolation of genomic DNA. In general,
all methods involve disruption and lysis of the cells followed by the removal of proteins and other
contaminants and finally, recovery of the DNA. Removal of proteins is typically done by digestion
with proteinase K, followed by the process like salting-out, organic extraction, or binding of the
DNA to a solid-phase support (either anion-exchange or silica technology). DNA is usually
recovered by precipitation using either ethanol or isopropanol. The choice of a method depends
on many factors such as the required quantity and molecular weight of the DNA and the purity
required for downstream applications of isolated DNA.
1.1 TOTAL DNA EXTRACTION FROM AGROBACTERIUM

TUMEFACIENS AND E. COLI
Protocol
1. Harvest the cells from 1.5 mL culture by centrifugation at 10,000 rpm, 4oC for
5 minutes.
2. Resuspend and lyse the cells in 200 mL of lysis buffer by vigorous pipetting.
3. To remove most proteins and cell debris, add 66 mL of 5 M NaCl and mix well, leave the
tube at –20oC for 10 min.
4. Centrifuge the viscous mixture at 12,000 rpm for 10 minutes at 4oC.
5. After transferring the clear supernatant to a fresh tube, equal volume of phenol:
chloroform and gently invert tube at least 50 times till a milky white solution is
completely formed. Centrifuge at 12,000 rpm for 3 minutes at 4oC.
6. Transfer the upper phase to a fresh tube and extract once with ether. The final lower
1
Laboratory Manual for Genetic Engineering
phase containing the DNA has to be collected and used for precipitation. Ether forms
the upper phase because of its less dense nature when compared to the cleared lysate.
7. Precipitate the DNA with 4 volumes of ice cold 100% ethanol. Wash 2 times with 70%
ethanol. Dry the DNA pellet under vacuum and redissolve in 50 mL of TE buffer.
8. This protocol also works well for the genera Xanthomonas, Pseudomonas and
Rhizobium.
Buffers
(i) Lysis buffer
Tris acetate – 400 mM
Sodium acetate – 20 mM
EDTA – 1 mM
SDS – 1%
(ii) 5 M NaCl
NaCl – 292.2 g
Water – 1 L
(iii) TE buffer
Tris–Cl – 10 mM
EDTA – 1 mM
Distilled water –1 L
pH 8
1.2 TOTAL CELL DNA ISOLATION FROM

BACILLUS THURINGIENSIS
This method makes use of lysozyme and SDS for lysing the bacterial cell wall and cell membrane
and sodium chloride for salting out of major proteins and cell debris. Phenol and chloroform are
used for removing traces of proteins leftover in the lysate after salting out step.
Protocol
1. Grow Bacillus thuringiensis single colony from nutrient agar plate in 5 mL Luria broth
with 0.1% glucose at 30oC at 250 rpm shaker.
2. Inoculate 1.5 mL overnight culture into 250 mL fresh LB with 0.1% glucose at 30oC and
grow up to 0.8–0.9 OD (for ~4 hours).
3. Harvest the cells by centrifugation at 10,000 rpm, 4oC for 10 minutes and wash the cells
once with washing solution.
4. Resuspend the cells in 5 mL resuspension solution containing 0.5 mg/mL lysozyme,
gently vortex and incubate at 37oC for 20 minutes.
5. Lyse the cells by adding 6.25 mL of lysis buffer. Mix gently by inversions for 3 – 4 times.
Incubate at 60oC for 30 minutes.
6. Centrifuge at 10,000 rpm, 4°C for 10 minutes. Carefully remove supernatant with a cut
blue tip and store the supernatant.
Isolation of Genomic DNA !
7. To the pellet add 2 mL of fresh lysis buffer mix gently and incubate at 60°C for
10 minutes. Spin and collect supernatant and pool it with the supernatant of previous step.
8. Add equal volume of equilibrated phenol to the cleared supernatant. Mix gently by
inversions. Spin and take the aqueous phase and add equal volume of chloroform:
Isoamyl alcohol (24:1) gently mix and repeat extraction for another 2 times and collect
the aqueous phase after centrifugation.
9. Add 4 volumes of ice cold absolute ethanol. Mix well and store at –70°C for 20 minutes
or 20°C for 1 hour.
10. Spin at 14,000 rpm, 10°C for 15 minutes and wash the pellet with 70% ethanol at
14,000 rpm, 10°C for 10 minutes. Air dry the pellet at 37°C or at room temperature.
11. Add 2.5 mL T10E1 mM (pH 8.0) and gently dissolve the pellet.
12. Add RNase A to a final concentration of 100 mg/mL. Mix incubate 37°C for 1 hour. Add
protease (100 mg/mL) and incubate 37°C for 1 hour.
13. Transfer the content to dialysis membrane. Dialysis at 4°C in 500 mL of 1X TE. Change
buffer once in 10 – 18 hours for 3 times.
14. Collect dialyzed sample and do phenol extraction for 2 times and chloroform: Isoamyl
alcohol for 2 times and take the final aqueous phase and add 0.1 volume of 3 M Sodium
acetate (pH 4.6) and add four volumes of ice cold absolute alcohol.
15. Store at –70°C for 20 minutes or 20°C for 1 hour. Spin and take the pellet and wash
once with 70% ethanol. Air dry the pellet and dissolve it in 200 mL of TE buffer.
Note:
This method is applicable to all the Bacilli strains.
Buffers
(i) Washing solution
NaCl – 100 mM
Tris – 10 mM
EDTA – 10 mM (Adjust pH 7.9 with conc. HCl)
(ii) Resuspension solution
NaCl – 150 mM
EDTA –100 mM
(Adjust pH 7.9 with conc. HCl)
(iii) Lysis buffer
Tris – 100 mM
NaCl – 100 mM
SDS – 2%
(iv) Phenol equilibrating buffer
Tris – 10 mM
NaCl – 100 mM
" Laboratory Manual for Genetic Engineering
(v) DNA dissolving and storage buffer

Tris – 10 mM
EDTA – 1 mM
(Adjust pH 7.5 – 8.0 with conc. HCl)
(vi) Neutral phenol preparation
Take dist. Phenol or water saturated phenol. Add 0.1% (w/v) of hydroxyquinoline and
dissolve. Then add equal volume of phenol equilibrating buffer. Shake well and allow it
to settle. Two phases will settle. Use the lower phase.
(vii) Buffered phenol/chloroform
Phenol (unbuffered) – 50 mL
Chloroform – 50 mL
Water – 20 mL
Hydroxyquinoline – 100 mg
(Mix well and use the lower phase)
(viii) RNase A
Stock solution: 10 mg/mL in water. Boil in boiling water bath for 10–15 minutes and
cool to room temperature and store at 4°C.
Working conc. 100 mg/mL
1.3 GENOMIC DNA ISOLATION FROM BLOOD

The blood samples (stored at 70°C in EDTA) are thawed, standard citrate buffer is added, mixed
and the tubes are centrifuged. The top portion of the supernatant is to be discarded and additional
buffer is added mixed, and again the tube is centrifuged. After the supernatant is discarded, the
pellet is resuspended in a solution of SDS detergent and proteinase K and the mixture are
incubated at 55°C for one hour. The sample is then to be phenol extracted, once with a phenol/
chloroform/isoamyl alcohol solution and after centrifugation, the aqueous layer is removed to a
fresh microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer, and then
ethanol is precipitated for the second time. Once the pellet is dried, the DNA can be resuspended
by TE buffer.
Protocol
1. Blood samples typically were obtained as 1 mL of whole blood stored in EDTA
vacutainer tubes frozen at –70°C.
2. Thaw the frozen samples, and to each 1 mL sample, add 0.8 mL 1X SSC buffer, and
mix. Centrifuge for 1 minute at 12,000 rpm in a micro-centrifuge.
3. Remove 1 mL of the supernatant and discard into disinfectant.
4. Add 1 mL of 1X SSC buffer, vortex and centrifuge as above for 1 minute and remove
all of the supernatant.
5. Add 375 mL of 0.2 M sodium acetate (NaOAc) buffer to each pellet and vortex briefly.
Then add 25 mL of 10% SDS and 5 mL of proteinase K (20 mg/mL in H2O), vortex briefly
and incubate for 1 hour at 55oC.
Isolation of Genomic DNA #
6. Add 120 mL phenol: chloroform/isoamyl alcohol (1:1) and vortex for 30 seconds.
Centrifuge the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.
7. Carefully remove the aqueous layer to a new 1.5 mL microcentrifuge tube, add 1 mL
of cold 100% ethanol, mix, and incubate for 15 minutes at 20°C.
8. Centrifuge for 2 minutes at 12,000 rpm in a micro-centrifuge. Decant the supernatant
completely.
9. Add 180 mL of TE buffer, vortex, and incubate at 55oC for 10 minutes.
10. Add 20 mL 2 M sodium acetate and mix. Add 500 mL of cold 100% ethanol, mix and
centrifuge for 1 minute at 12,000 rpm in a micro-centrifuge.
11. Decant the supernatant and rinse the pellet with 1 mL of 80% ethanol. Centrifuge for
1 minute at 12,000 rpm in a micro-centrifuge.
12. Decant the supernatant, and dry the pellet for 10 minutes.
13. Resuspend the pellet by adding 200 mL of TE 10:1(10 mM Tris and 1 mM EDTA) buffer.
Incubate overnight at 55oC, vortexing periodically to dissolve the genomic DNA. Store
the samples at –20oC.
Buffer
(i) 1X SSC buffer
Sodium chloride – 150 mM
Sodium citrate – 5 mM
Adjust pH to 7.0 with NaOH
1.4 A RAPID PROCEDURE FOR ISOLATION OF

DNA CULTURED MAMMALIAN CELLS
Protocol
1. Resuspend the cultured mammalian cells at 107/mL in T10E10 buffer.
2. Add SDS and proteinase K to a final concentration of 0.5% and 200 µg/mL respectively.
3. Mix and incubate at 55oC for 2 hours.
4. Add NaCl to a final conc. of 0.2 M.
5. Extract twice with equal volume of phenol: chloroform and once with chloroform.
6. Place the tube with cap open in 55oC water bath for 1 hour.
7. Add RNase (25 µg/mL) and incubate for 1 hour at 37oC.
8. Extract once with phenol: chloroform and once with chloroform.
9. Precipitate DNA with 1–2 volumes of ice cold ethanol. Incubate at 20oC for 1 hour.
10. Pellet DNA by centrifuging at 12,000 rpm for 10 minutes.
11. Resuspend the air dried DNA pellet in TE buffer.
Buffers
(i) 10 % SDS
SDS – 10 g
Water – 100 mL
$ Laboratory Manual for Genetic Engineering
(ii) 2 M NaCl
NaCl– 116.8 g
Water– 1 L
(iii) TE buffer
Tris–Cl – 10 mM
EDTA – 10 mM
1 L distilled water (adjust pH 8)
REFERENCES
James, W., Y.F. Chan and H. Paul Goodwin (1995), Extraction of Genomic DNA from
Extracellular Polysaccharide-Synthesizing Gram-Negative Bacteria, BioTechniques,
18 (3), pp. 418–422.
Laird, P.W., A. Zijderveld, K. Linders, M.A. Rudnicki, R. Jaenisch and A. Berns (1991), Nucleic
Acids Res., 19 (15), p. 4293.
Rudbeck, L. and J. Dissing (1998), Rapid, simple alkaline extraction of human genomic DNA
from whole blood, buccal epithelial cells, semen and forensic stains for PCR, Biotechniques,
25, pp. 588–592.
Sambrook, J. and D.W. Russell (2001), Molecular Cloning: A Laboratory Manual, 3rd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 1.31–1.38.
Sharma, R.C., A.J.M. Murphy, M.G. DeWald and R.T. Schimke (1993), A rapid procedure for
isolation of RNA-free genomuic DNA from mammalian cells, BioTechniques, 14,
pp. 176–178.
Tuli, R., J. Saluja and N.K. Notani (1989), Cloning and expression in Escherichia coli of
entomotoxic protein gene from Bacillus thuringiensis subsp., kurstaki. J. Genet, 68,
pp. 147–160.
2
Isolation of Plasmid DNA from GRAM
Negative and GRAM Positive Bacteria
A large number of methods have been developed to purify plasmids from bacteria. These
methods include the following steps:
• Growth of bacteria
• Harvesting and lysing of bacteria and
• Purification of the plasmid DNA
2.1 GROWTH OF BACTERIA

Plasmid DNA should be purified from a culture inoculated with a single bacterial colony having
plasmid molecule which is grown to log phase. This culture may be used for the preparation of
plasmids in a mini preparation or a large-scale preparation. The culture used for the preparation
of plasmid is usually grown in a selective condition, i.e., in the presence of an appropriate
antibiotics in the medium (Table 2.1).
TABLE 2.1 Plasmid growth and replication

Replicon Copy number Low copy/ Comments
High copy
PMB1 or Several High copy or pUC plasmids have replicons derived from
pUC derivative hundred relaxed pMB1 and replicate to a hundreds of copies.
These plasmids can be further amplified to
thousands of copies by the addition of
chloramphenicol (170 mg/mL) during log
phase of growth and growing further 8 hours
with vigorous shaking.
Col E1 (pBR322) 15 High copy or These plasmids maintain a low moderate
relaxed copy number in transformed cells. This
could be increased by the addition of
7 (Contd.)
& Laboratory Manual for Genetic Engineering
TABLE 2.1 Plasmid growth and replication (Contd.)

Replicon Copy number Low copy/ Comments
High copy
chloramphenicol (170 mg/mL) during log
phase of growth and growing further 8 hours
with vigorous shaking.
pSC101 5 Low copy or It is not easy to grow low copy number plas-
stringent mids. The copy number of these plasmids
can be reportedly increased by growing the
cells in terrific broth.
2.2 HARVESTING AND LYSING OF BACTERIA

Harvesting of bacterial cells for DNA isolation is done by centrifugation and followed by lysing
of cells. The lysing of cells can be done by any one of a large number of treatments with either
non-ionic or ionic detergents, organic solvents, alkali or heat. The heat treatment for lysing of
cells is not ideal when DNA is to be isolated from E. coli strain HB 101, which releases large
carbohydrates as well as this strain, is having endonuclease A which cannot be inactivated by
boiling procedures.
For plasmids of large sizes ≥ 15 kb should be handled with great care and gentleness during
lysis. Gentle lysis is best accomplished by suspending the bacteria in an iso-osmotic solution of
sucrose and treating them with lysozyme and ethylenediaminetetraacetic acid (EDTA), which
removes most of the cell wall materials during treatment. The spheroplasts are then lysed
completely by the addition of an anionic detergent such as sodium dodecyl sulphate (SDS).
For smaller plasmids more severe methods can be used, and no special care needs to be taken
to minimize the shearing forces. It could be very well exposed to detergent and lysed by boiling or
treating with alkali. This leads to denaturing of linear chromosomal DNA of the host whereas, the
super helical plasmid DNA are unable to separate from each other because they are topologically
intertwined. At neutral pH, the plasmid molecules are able to form a super coiled form.
2.3 PURIFICATION OF THE PLASMID DNA

The plasmid preparations following the above method will yield plasmid DNAs mostly
contaminated with both RNA and chromosomal DNA of the host. These DNA preparations could
be very well used as substrates for the restriction digestion and for DNA synthesis using DNA
polymerases. The contaminating molecules must be removed before they are used for gene
cloning or for transferring into mammalian cells.
The most efficient method of separating supercoiled plasmid DNA from the contaminating
fragments of bacterial DNA is the buoyant density gradient centrifugation in cesium chloride-
ethidium bromide (Clewell and Helinski,1969). The separation depends on the differences in the
binding of ethidium bromide molecules into linear DNA molecules and covalently closed circular
plasmid DNA molecules. Linear DNA molecules allow the binding of ethidium bromide more
strongly than the ccc plasmid DNA because of their free ends. At the same time ccc plasmid
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria '
DNAs are having no free ends to allow the binding of more ethidium bromide molecules and
hence minimum binding of ethidium bromide takes place in ccc plasmid DNA. More binding of
ethidium bromide to linear double stranded DNA reduces their buoyant density in CsCl solution
(1.54 g/cm3) and hence they form a band just above the ccc plasmid band (whose buoyant
density is 1.59 g/cm3).
2.3.1 Cloning in Plasmid Vectors

Cloning in plasmid vectors is an easy and straightforward process. The plasmid molecule is
digested with one restriction enzyme having unique cleavage site and ligated with a foreign DNA
having compatible ends. The ligated DNA are then transformed into an appropriate E. coli strain and
the resulting transformants are screened by any one of the screening and selection techniques such
as hybridization, restriction mapping or PCR to identify the presence of desired DNA sequences.
2.3.2 Cloning DNA Fragments with Protruding Ends

The easiest DNA fragments to be cloned are with either 5¢ or 3¢ overhanging ends. These single
stranded overhanging ends of 1–6 bp in length are created by digestion of the vector as well as
the target DNA with restriction enzymes that cuts asymmetrically within the recognition
sequences. When the ends protruding from the DNA fragments are similar or compatible, they
can anneal to form a linear hybrid molecule that can be stabilized by a DNA ligation by DNA ligase
that repairs the broken phosphodiester bonds in between nucleotides to form a circular
recombinant plasmid capable of transforming E. coli.
The circular plasmids can have the foreign DNA fragment inserted in either orientation in the
case of insert DNAs with similar ends that are created by the cleavage of single restriction
enzyme [Figure 2.1(a)]. If the DNA ends created by the cleavage of double restriction
endonucleases with different recognition sequences, the ends of DNA fragments are dissimilar
and unable to ligate each other. The foreign DNA will ligate only to a plasmid DNA cleaved with
the same two enzymes, generating a high yield of circular recombinants containing a single insert
in a predefined orientation. This cloning process is sometimes called as directional cloning or
forced ligation [Figure 2.1(b)]. After cloning, the recombinant plasmid is transformed into
competent bacterial cell and the clone is selected by antibiotic markers [Figure 2.1(c)].
2.4 MODIFIED ALKALINE LYSIS PROTOCOL FOR BOTH

E. COLI AND BACILLUS SP.
Principle
Alkaline lysis is the method of choice for isolating circular plasmid DNA from bacterial cells. It
is probably one of the most generally useful techniques as it is a fast, reliable and relatively clean
way to obtain DNA from cells. If necessary, DNA from alkaline lysis preparation can be further
purified. The alkaline lysis method is based on a unique property of plasmid DNA. It is able to
rapidly anneal following denaturation. This allows the plasmid DNA to be separated from the
bacterial chromosome.
Typically, E. coli cells having the plasmid are to be grown in a rich medium for isolation of
plasmid DNA. Then the cells are to be lysed with a detergent and an alkali mixture and the plasmid
DNA are to be isolated. The cell debris is precipitated using SDS and potassium acetate.
Isopropanol is then used to precipitate the plasmid DNA from the supernatant and the DNA is
resuspended in DNA dissolving and storage buffer (often TE buffer). A mini prep method usually
yields 5–10 mg. This can be scaled up to midi prep or a maxi prep, which will yield much larger
amounts of DNA.
Foreign
HindIII DNA
Foreign Region of
DNA HindIII interest
Region of Gene for
Gene for antibiotic Plasmid
antibiotic Plasmid EcoRI interest EcoRI
resistance EcoRI
resistance EcoRI
EcoRI HindIII EcoRI HindIII EcoRI
EcoRI EcoRI
Sticky ends
Sticky ends
Hybridization
Hybridization +DNA ligase
+DNA ligase Recombinant
Recombinant DNA
DNA
Figure 2.1(a) General cloning method. Figure 2.1(b) Directional cloning method.
Recombinant
DNA
Transformation
Recombinant
Bacterial Bacteria platted on
plasmid
chromosome medium + antibiotics
Cell division
Only bacteria containing
recombinant DNA grow
Culture
DNA
purification
Figure 2.1(c) Transformation of recombinant DNA in bacterial cell and clone selection.
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria
Protocol
1. Pellet the E. coli/Bacillus sp. cells from 50 mL bacterial culture (log phase) and
resuspend the cells in 1 mL of TEG (pH 8.0).
2. Add 5 mg/mL lysozyme for Bacillus sp. and (0.05 mg/mL for E. coli) and keep at 37oC
for 30 minutes to 1 hour for Bacillus sp. and 10 minutes at room temperature for E. coli.
3. Add 1 + 1 mL of 1% SDS and add 0.2 N NaOH. Mix well and keep it in ice for
10 minutes.
4. Add 1 mL of ice cold 3 M sodium acetate (pH 5.2). Mix well and add 400 mL of
TE (pH 8.0) and leave the tubes in the - 20oC freezer for overnight or for 24 hours.
5. Centrifuge and take the supernatant and extract once with equilibrated phenol and
chloroform.
6. Precipitate the DNA with 2 volumes of ice cold ethanol and centrifuge at 10,000 rpm
for 10 minutes at 10oC, collect the plasmid DNA pellet and wash the DNA pellet with
70% ethanol and air dry the final DNA pellet and resuspend the DNA in 1 mL of TE
buffer.
Buffers
(i) TEG buffer (pH 8.0)
Tris – 25 mM
EDTA – 10 mM
Glucose – 50 mM
(ii) 0.2 N NaOH and 1% SDS buffer (100 mL)
0.8 g in 100 mL
1% SDS
1.0 g in 100 mL
(iii) 3 M Sodium acetate (pH 4.6 or 5.2)
(Dissolve sodium acetate salt in least amount of H2O and adjust pH 4.6 or 5.2 by glacial
acetic acid).
2.5 HIGH MOLECULAR WEIGHT PLASMID PREPARATION

FROM GRAM NEGATIVE BACTERIA
(INCLUDING AGROBACTERIUM TUMEFACIENS)
Protocol
1. Inoculate 0.25 mL of overnight culture of Agrobacterium tumefaciens cells harbouring
Ti-plasmid into 5 mL of Luria broth and shake the culture at 29oC for 6 hours.
2. Take 1 mL of log phase culture and vortex with equal volume of ethanol-phenol solution.
3. Harvest the cells by centrifugation and resuspend them in 200 mL of 20 mM Tris
HCl (pH 8.0), 10 mM EDTA buffer.
4. Transfer the cell suspension to an Eppendorf tube. Add lysozyme 0.5 mg/mL; 20 mL of
20% SDS incubate at 37oC for 5 minutes and then centrifuge for 15 minutes at 4oC in
an Eppendorf at 12,000 rpm.
5. Collect the supernatant and check 10 mL of the plasmid preparation on a 0.7% agarose
gel.
Buffers
(i) Ethanol-phenol solution
Ethanol – 75%
Phenol (Equilibrated) – 2%
(ii) 20% SDS
SDS – 20 g
Water – 100 mL
2.6 PLASMID ISOLATION FROM E. COLI (MINI PREPARATION)

Alkaline lysis in combination with the detergent SDS has been used for more than 40 years to
isolate plasmid DNA from E. coli. Exposure of bacterial cell suspensions to the strongly anionic
detergent at high alkaline pH opens the cell wall, denatures chromosomal DNA and proteins and
releases plasmid DNA into the supernatant. Although the alkaline solution completely disturbs
base pairing, the strands of closed circular plasmid DNA are unable to separate from each other,
because they are topologically intertwined. As long as the intensity and duration of exposure to
alkali is not too great, two strands of plasmid DNA fall once again into the regular conformation
when the pH is returned to neutral.
During lysis bacterial proteins, broken cell walls and denatured chromosomal DNA become
enmeshed in large complexes that are coated with dodecyl sulphate. These complexes are
efficiently precipitated in solution than sodium ions are replaced by potassium ions. After the
denatured material has been removed by centrifugation, native plasmid DNA can be recovered
from the supernatant. Alkaline lysis in the presence of SDS is flexible technique that works well
with all strains of E. coli and with bacterial cultures ranging in size from 1 mL to > 500 mL.
The covalently closed circular plasmid DNA recovered from the lysate can be purified in many
different ways and to different extents, according to the needs of the experimenter.
Protocol
1. Resuspend the pellet from 1.5 mL bacterial culture in 100 mL of TEG buffer.
2. Add 200 mL of 0.2 N NaOH and 1% SDS buffer. Mix gently till all cells get lysed
completely.
3. Add 150 mL of 3 M sodium acetate (pH 4.6 or 5.2), mix gently and incubate at - 20oC
for 10 minutes and centrifuge at 15,000 rpm for 10 minutes at 4oC. Transfer the cleared
supernatant to another sterile tube.
4. Add 720 mL of isopropanol to the supernatant, mix gently and centrifuge for 15 minutes
at 15,000 rpm at 10oC.
5. Wash the DNA pellet with 70% ethanol.
6. Resuspend the pellet in 100 mL of TE buffer.
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria !
7. Add 100 mg of cesium chloride (CsCl)

shake and dissolve. Add 10 mL of ethidium
Wells
bromide from 10 mg/mL stock. Mix com-
pletely and centrifuge for 2 minutes at
10,000 rpm. Carefully remove the superna-
tant and extract ethidium bromide using
50 mL of isobutanol per extraction for
3 times.
8. To the cleared CsCl DNA solution add
Plasmid DNA
400 mL TE buffer and then add 50 mL of
3 M NaOAc (pH 4.6) and 720 mL of
isopropanol. Mix and centrifuge for
5 minutes at 10,000 rpm. Figure 2.2 Agarose gel electrophoresis
9. Wash DNA pellet in 70% ethanol, air dry the of plasmid DNA.
DNA pellet for 2 minutes and resuspend in 20 mL of TE buffer and check it on an agarose
gel (Figure 2.2).
Buffers
(i) TEG (pH 8.0)
Tris – 25 mM
EDTA – 10 mM
Glucose – 50 mM
(ii) NaOH/SDS
NaOH – 0.2 N
SDS – 1%
(iii) 3 M Sodium acetate (pH 4.6)
(iv) TE buffer (pH 8.0)
Tris – 10 mM
EDTA – 1 mM
2.7 LARGE SCALE PLASMID PREPARATION FOR

E. COLI (ALKALINE LYSIS)
The method used for the isolation of large scale cosmid and plasmid DNA is a modification of
an alkaline lysis procedure. Briefly, cells containing the desired plasmid or cosmid are harvested
by centrifugation, incubated in a lysozyme buffer, and treated with alkaline detergent. Detergent
solubilized proteins and membranes are precipitated with sodium acetate, and the lysate is cleared
first by filtration of precipitate through cheese cloth and then by centrifugation. The DNA-
containing supernatant is transferred to a new sterile tube and the plasmid or cosmid DNA is
precipitated by the addition of either polyethylene glycol/or by isopropanol/or by ice cold 100%
ethanol and collected by centrifugation.
2.7.1 Plasmid Amplification

1. Incubate 10 mL of LB with appropriate antibiotic with a single bacterial colony. Incubate
at 37oC O/N with vigorous shaking.
2. Subculture 25 mL of LB in a 100 mL flask with appropriate antibiotics with 0.1 mL of
O/N culture. Incubate at 37oC till it reaches late log phase (OD600 = 0.6).
3. Incubate 25 mL of the late log phase culture into 500 mL of LB prewarmed to 37oC with
appropriate antibiotics in a 2 L flask. Incubate exactly 2.5 hours at 37oC with vigorous
shaking. The OD600 of the culture will be approximately 0.4.
4. Add 2.5 mL of a solution of chloramphenicol (34 mg/mL in ethanol). The final
concentration of chloramphenicol in the culture should be 170 mg/mL.
5. Incubate at 37oC with vigorous shaking for a further 12–16 hours.
2.7.2 Harvesting
Harvest the bacterial cells by centrifugation at 4000X g for 10 minutes at 4oC. Discard the
supernatant. Wash in 100 mL of STE buffer (0.1 M NaCl; 10 mM Tris-Cl pH 7.8 and 1 mM
EDTA) or with TEG buffer.
2.7.3 Lysis with Alkali

1. Resuspend the bacterial cell pellet from 200 mL culture in 10 mL of TEG buffer
containing 5 mg/mL of lysozyme. Let stand at room temperature for 5 minutes.
2. Add 20 mL of freshly made NaOH/SDS buffer and mix the contents gently by inversions
several times and incubate in ice for 10 minutes.
3. Add 15 mL of an ice-cold solution of 3 M potassium acetate or sodium acetate (pH 4.6).
Mix the contents gently by inversions, several times. Let stand on ice for 10 minutes.
4. Centrifuge at 12,000 rpm for 15 minutes at 4oC. The chromosomal DNA and the
bacterial debris should form a tight pellet at the bottom of the tube.
5. Transfer the clean supernatant into a clean tube and add 0.7 volume of isopropanol. Mix
well and let stand at room temperature for 15 minutes.
6. Recover the plasmid DNA by centrifugation at 12,000 rpm for 30 minutes at 10oC (salt
may be precipitated if centrifugation is carried out at 4oC).
7. Discard the supernatant. Wash the pellet with 70% ethanol at room temperature.
8. Dry the pellet and resuspend the DNA in TE (pH 8.0) buffer and check on a 0.7%
Agarose gel.
2.8 RAPID BOILING METHOD OF ISOLATION OF

BACTERIAL PLASMID
Protocol
1. Grow the E. coli cells bearing plasmid to log phase and centrifuge 1.5 mL of culture in
a microfuge tube for 1–2 minutes.
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria #
2. Discard the supernatant and resuspend the cell pellet in 1 mL of TNE buffer.
3. Repeat centrifugation, discard the supernatant and resuspend the washed cell pellet in
100 mL of TNE buffer containing 2 mg/mL lysozyme.
4. Incubate 15–20 minutes at room temperature and place the tube in boiling water both
for 1 min. Cool rapidly on ice by plunging suddenly the tube in ice. Keep it in ice for
5 more minutes.
5. Centrifuge the microfuge tube using a refrigerated centrifuge for 10 minutes at
10, 000 rpm at 10oC.
6. Collect the plasmid DNA preparation that is nothing but the supernatant and check an
aliquot on an agarose gel.
Buffer
(i) TNE buffer (pH 8.0)
Tris HCl – 10 mM
NaCl – 100 mM
EDTA – 1 mM
2.9 ISOLATION OF PLASMID DNA FROM

BACILLUS THURINGIENSIS
Protocol
1. Incubate single colony into 5 mL nutrient broth. Grow it overnight in a rotary shaker at
160 rpm.
2. Subculture 0.5 mL into 50 mL nutrient broth and grow it up to 0.6 OD to 0.8 OD at
600 nm.
3. Pellet the cell from 50 mL culture and lyse with 8.5 mL of T50 mM and E20 mM buffer
(pH 8.5) containing 2 mg of lysozyme per mL, 0.5 mL of 20% SDS solution and
1.0 mL of a 5 mg/mL solution of protease in above TE buffer.
4. After mixing with gentle inversions, the suspension should be incubated at 37oC for
30 minutes.
5. Subsequently, 0.3 mL of 3 N NaOH was added to the suspension and mixed gently for
3 minutes.
6. Neutralize the suspension by the addition of 0.6 mL in 2 M Tris-HCl (pH 7.0) and mixed
gently.
7. Add 1 mL of 5 M NaCl to the suspension and mix by inversions and place on ice for
15 minutes and then centrifuge at 12,000 rpm for 15 minutes.
8. Precipitate the DNA from the supernatant with ethanol and keep at - 20oC for 1–5 hours
and centrifuge.
9. Discard the supernatant and air dry the pellet by inverting the tube on a paper towel for
a few minutes.
10. Dissolve the DNA pellet in 1 mL of T10E1 buffer (pH 8.0).
Buffers
(i) T50 mM E20 mM buffer (pH 8.5)
Tris – 50 mM
EDTA – 20 mM
(ii) 20% SDS (100 mL)
SDS – 20 g
(iii) 3 N NaOH
(iv) 2 M Tris – HCl (pH 7.0)
(v) 5 M NaCl
(vi) TE buffer (pH 8.0)
Tris – 10 mM
EDTA – 1 mM
2.10 PURIFICATION OF HIGH MOLECULAR WEIGHT PLASMIDS

FROM BACILLUS THURINGIENSIS
Protocol
1. Grow cultures in 500 mL Luria broth supplemented with 0.1% yeast extract and 0.1%
glucose at 30oC.
2. Harvest the cells at an O.D of 0.8 at 600 nm.
3. Resuspend the cells in 5 mL of 0.05 M Tris and 0.02 M EDTA (pH 7.9).
4. Lyse the cells by adding 95 mL of TE containing 1% SDS and 0.085 M NaOH (pH 12.4).
After 30 minutes at room temperature with occasional mixing, add 10 mL of 10% SDS
and mix the solution gently.
5. Add 10 mL of 2 M Tris (pH 7.0) and also add 30 mL of 5 M NaCl and store the
preparation at 4oC overnight.
6. The mixture can then be centrifuged at 11,000 for 15 minutes. Take the supernatant.
7. Add RNase at a final concentration of 2 mg/mL and 36 mL of 50% PEG6000.
8. Leave the preparation in ice for at least 2 hours.
9. Plasmids can be obtained by centrifugation at 12,000 for 15 minutes at 4oC. Collect the
pellet, wash it with 70% ethanol and air dry.
10. Resuspend the pellet in T10E1 mM buffer (pH 8.0).
Buffers
(i) T10E1 buffer (pH 8.0)
Tris-HCl– 10 mM
EDTA – 1 mM
(ii) 5 M Sodium chloride
NaCl – 292.2 g
Water – 1000 mL
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria %
2.11 ISOLATION OF PLASMID DNA FROM BACILLUS SUBTILIS AND

BACILLUS MEGATERIUM
Protocol
1. Inoculate a loopful of bacterial culture (B. megaterium and B. subtilis) in 5 mL of Luria
broth and grow at 37oC shaking for overnight.
2. Subculture 1 mL of overnight culture into 100 mL fresh Luria broth and grown at 37oC
for 4 hours with vigorous shaking.
3. Harvest the cells at 10,000 at 5oC for 15 minutes.
4. Wash the cells with 1X TES buffer.
5. Resuspend the cells with 10 mL of lysozyme mix containing 1 mg/mL lysozyme and
keep at 37oC for 30 minutes.
6. Lyse the cells with 0.2% SDS, mix gently and incubate the mixture at 50oC for 15 minutes.
7. And then add 5 mL of 5 M NaCl mix and incubate at 50oC for 15 minutes.
8. Leave the tubes in ice for 30 minutes and then centrifuge at 10,000 rpm in 5oC for 10
minutes.
9. Carefully remove the supernatant and add equal volume of neutral phenol. Mix and
centrifuge at 10, 000 rpm at 5oC for 10 minutes.
10. Collect the aqueous phase. To which add equal volume of CHCl3: IAA (24:1). Mix
thoroughly and centrifuge at 10,000 rpm at 5oC for 10 minutes.
11. Remove supernatant and add 3 volume of ice cold absolute ethanol. Mix well and
incubate the tubes at -20o for overnight.
12. Centrifuge at 10,000 rpm at 5oC for 15 minutes and collect the pellet, wash it with 70%
ethanol and dry the pellet. Resuspend the pellet in T10E1 buffer.
Buffers
(i) TES buffer (pH 8.0)
Trizma base – 30 mM
Na EDTA – 5 mM
NaCl – 50 mM
(ii) 2% SDS
(iii) 5 M NaCl
(iv) TE buffer (8.0)
Tris – 10 mM
EDTA – 1 mM
2.12 PURIFICATION OF GENOMIC AND PLASMID DNA THROUGH

PHENOL CHLOROFORM TREATMENT
Protocol
1. Dilute the isolated DNA in TE buffer up to 500 mL.
2. Add 500 mL of water saturated/equilibrated phenol. Mix well and centrifuge at 5000 rpm
for 5 minutes at 4oC.
3. Transfer the upper aqueous phase to another sterile Eppendorf.

4. Add 500 mL of chloroform and isoamyl alcohol (24:1). Mix well and centrifuge at
5000 rpm for 5 minutes.
5. Transfer the upper layer to another eppendorf and add sodium acetate (pH 4.6) to the
final concentration of 0.3 M. Mix well and add one volume of isopropanol.
6. Mix well and incubate at room temperature for 10 minutes and centrifuge at 10,000 rpm
for 10 minutes at 10oC.
7. Decant the supernatant and wash the DNA pellet with 200 mL of 70% ethanol and
centrifuge at 10,000 rpm for 7 minutes at 10oC.
8. Remove 70% ethanol by pipetting and air dry the pellet and resuspend the pellet in
200 mL of TE buffer.
2.13 ULTRA PURIFICATION OF PLASMID DNA THROUGH CESIUM

CHLORIDE ETHIDIUM BROMIDE GRADIENT
Separation of plasmid and chromosomal DNA by buoyant density centrifugation gradients
containing cesium chloride (CsCl) and ethidium bromide depends on the difference between the
amounts of ethidium bromide that can bind to linear and closed circular DNA molecules.
For many years, equilibrium centrifugation in CsCl-ethidium bromide gradients was the
method of choice to prepare large amounts of plasmid DNA. However, this process is expensive
and time consuming so many alternative methods have been developed, including differential
precipitation and column chromatography. Nevertheless, traditionalists of whom there are many,
still believe the plasmids purified by banding in CsCl-ethidium bromide gradients are the purest
and best DNAs for molecular biological experiments. Covalently closed circular (ccc) DNAs
prepared by isopycnic centrifugation in CsCl-ethidium bromide gradients are contaminated by
small fragments of DNA and RNA derived from the host bacteria. These small fragments take
far longer to reach equilibrium in CsCl-ethidium bromide gradients, than the larger plasmid DNAs.
Hence, when molecules of covalently closed circular DNA are at equilibrium, small fragments
are still fairly evenly distributed throughout the gradient. This problem can be solved by
abandoning CsCl-ethidium bromide and purifying plasmids by chromatography on commercial
level, or it can be alleviated by subjecting closed circular DNA recovered from CsCl-ethidium
bromide to a second of equilibrium centrifugation.
Protocol
1. Add 4.4 gm of CsCl to 4 mL of DNA in TE buffer. Keep in cold room for 1 hour and
centrifuge at 10,000 rpm, 4oC for 10 minutes. In case of any pellet or clumping, remove
the clumping from the cleared supernatant carefully.
2. To the above clear DNA-CsCl solution and add 400 mL of ethidium bromide (10 mg/mL
stock).
3. Keep this solution in the dark room for 30 minutes and then load in 5 mL quick seal ultra
centrifuge tube.
4. Seal the tube properly using a sealer and centrifuge at 65,000 rpm at 20oC for 16 hours.
5. Take the tubes out of the rotor at the end of the run [Figure 2.3(a)]; insert a 20 gauge
needle into the top of the tube after visualizing the plasmid band and chromosomal bands
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria '
under UV illumination (usually plasmid band will be lower to the chromosomal band) as
shown in Figure 2.3(b).
Protein
precipitate
Increasing
concentration of Linear chromosomal
CsCl DNA
Circular plasmid
DNA
RNA pellet
Figure 2.3(a) Separation of circular plasmid DNA from linear chromosomal DNA after
CsCl – ethidium bromide density gradient centrifugation.
Figure 2.3(b) Recovery of plasmid DNA band by inserting a hypodermic syringe.
6. Gently recover the plasmid band using a 3 mL syringe from the sides of the tube fitted
with a 20 gauge needle [Figure 2.3(c)].
Recovered plasmid DNA in CsCl
Figure 2.3(c) Recovered circular plasmid DNA in a hypodermic syringe.

7. Add equal volume of isopropanol or butanol saturated with aqueous 5 M NaCl +10 mM
Tris and 1 mM EDTA (pH 8.5 or 8.0). Mix well and remove the top organic phase
containing ethidium bromide and repeat extractions until the top phase is colourless (The
number of extractions required is greater with more concentrated DNA).
8. To the cleared CsCl –DNA solution add 4 volumes of TE buffer or sterile distilled water
and 6 volumes of ice cold 100% ethanol and keep for precipitation in - 20oC incubator
for 24 hours.
9. Pellet the DNA by centrifugation at 10,000 rpm for 10 minutes at 10oC. Wash the pellet
with 70% ethanol and air dry the pellet.
10. Dissolve the DNA pellet in TE buffer (less volume based on the pellet size).
Note
CsCl usually 1 gm for 1 mL of DNA is needed.
Ethidium bromide at a final concentration of 600 mg/mL is needed.
Buffers
(i) TE saturated isopropanol
1. Take 50 mL of TE containing 5 M NaCl and add 100 mL of isopropanol/butanol.
Mix thoroughly.
2. Add small quantities of isopropanol successively till two layers are separated. Both
layers should be clear. The lower aqueous layer should have precipitated salt (this
indicates the saturation of isopropanol and aqueous layers). If this is not there, add
TE containing 5 M NaCl till the precipitated salt is seen.
(ii) TE containing 5 M NaCl
Take 5 mL of 10X TE (pH 8.0) and 45 mL of 5 M NaCl. Mix well.
(iii) 10X TE buffer (100 mL)
Trizma base – 100 mM (1.211 g)
EDTA – 10 mM (0.3722 g)
(iv) 5 M NaCl (100 mL)
NaCl – 29.2 g
REFERENCES
Birboin and Doly (1979), A rapid alkaline extraction procedure for screening recombinant plasmid
DNA, Nucleic acid research, 7, pp. 151–153.
Birnboim, H.C. and J. Doly (1979), A rapid alkaline procedure for screening recombinant plasmid
DNA, Nucleic Acids Res., 7, pp. 1513–1523.
Clewell, D.B. and D.R. Helinski (1969), Supercoiled circular DNA-protein complex in
Escherichia coli: purification and induced conversion to an open circular DNA form, Proc.
Nat. Acad. Sci., US, 62, pp. 1159–66.
Domenico, P., J.L. Marx, P.E. Schoch and B.A. Cunha (1992), Rapid plasmid DNA isolation from
mucoid gram-negative bacteria, J Clin Microbiol, 30 (11), pp. 2859–2863.
Isolation of Plasmid DNA from GRAM Negative and GRAM Positive Bacteria
Guerry, P., D.J. LeBlanc and S. Falkow (1973), General method for the isolation of plasmid
deoxyribonucleic acid, J. Bacteriol, 116, pp. 1064–1066.
Heierson, A., R. Landen, A. Lovgren, G. Dalhammar and H.G. Boman (1987), Transformation
of Vegetative Cells of Bacillus thuringiensis by Plasmid DNA, J. Bacteriol, 169(3), pp. 1147–
1152.
Ish-Horowicz, D. and J.F. Burke (1981), Rapid and efficient cosmid cloning, Nucleic Acids Res.,
9, pp. 2989–2998.
Plant Molecular Biology Reports (1983), 1(1), pp. 39–40.
Ramírez, A.R. and J.E. Ibarra (2008), Plasmid Patterns of Bacillus thuringiensis Type Strains,
Appl Environ Microbiol, 74(1), pp. 125–129.
Scheelf, M. and P. Heimann (1993), Cesium chloride or column preparation, An electron
microscopical view of plasmid preparations, Biotechniques, 14, p. 544.
Voskuil, M.I. and G.H. Chambliss (1993), Rapid Isolation and Sequencing of Purified Plasmid
DNA from Bacillus subtilis, Appl. Environ. Microbiol, 59(4), pp. 1138–1142.
22 Laboratory Manual for Genetic Engineering
3
Isolation of RNA from Bacteria and
Cultured Mammalian Cells
Purification of intact RNA from cells and tissues should be done very quickly to protect the RNA
molecules from cellular RNases. One may also deactivate cellular RNases during the RNA
purification process. Because of the urgency, many methods for the isolation of intact RNA from
cells use strong denaturants such as guanidinium hydrochloride or guanidinium thiocyanate to
disrupt cells, solubilize their components and denature endogenous RNases simultaneously.
The use of guanidinium isothiocyanate in RNA extraction, first mentioned briefly by Ullrich
et al., (1977), was documented in papers published by Han et al., (1987) and Chirgwin et al., (1979).
The Han method is cumbersome as it involves solubilization of RNA pellets in progressively
smaller volumes of 5 M guanidine thiocyanate. In the Chirgwin method, cultured cells or tissues
are homogenized in 4 M guanidine isothiocyanate and the lysate is layered onto a dense cushion
of CsCl. Because the buoyant density of RNA in CsCl (1.8 g/mL) is much greater than that of
other cellular components, rRNAs and mRNAs migrate to the bottom of the tube during
ultracentrifugation. As long as the step gradients are not overloaded, proteins remain in the
guanidinium lysate while DNA floats on the CsCl cushion. Because the Chirgwin method (1979)
yields RNA of very high quality and purity and is not labour-intensive, it became the standard
technique during the early 1980s for isolation of undegraded high molecular weight RNA.
However, the method has one weakness: it is unsuitable for simultaneous processing of many
samples. For this purpose, it has been almost completely displaced by the single step technique
of Chomczynski and Sacchi (1987), in which the guanidinium thiocyanate homogenate is
extracted with phenol: chloroform at reduced pH. Elimination of the ultracentrifugation step
allows many samples to be processed simultaneously and speedily at modest cost and without
sacrifice in yield or quantity of RNA.
There are two circumstances in which the single step procedure is now recommended. First,
the procedure does not extract RNA efficiently from adipose tissues that are rich triglycerides.
RNA is best prepared from these fatty sources by a modification of the method of Chirgwin
et al., (1979). Second, RNA prepared by guanidine lysis is sometimes contaminated to a significant
extent by cellular polysaccharides and proteoglycans. These contaminants are reported to prevent
solubilization of RNA after precipitation with alcohols, to inhibit Reverse Transcriptase–
22
Isolation of RNA from Bacteria and Cultured Mammalian Cells 23
Polymerase Chain Reactions (RT-PCRs), and to bind to membranes during RNA blotting. If
contamination by proteoglycans and polysaccharides appears to be a problem, include an organic
extraction step and change the conditions used to precipitate the RNA.
3.1 ISOLATION OF BACTERIAL RNA

Protocol
1. Grow bacteria in a minimal medium to an optical density of 0.5 to 0.6 at 600 nm.
2. Transfer 15 mL of bacterial culture portion to a pre-chilled 125-mL flask and centrifuge
the culture at 12,000X g at 0°C for 5 minutes.
3. Resuspended the cell pellet in 3 mL of a solution containing 0.02 M sodium acetate
(pH 5.2), 1 mM EDTA, and 0.2% diethylpyrocarbonate, and then sodium dodecyl
sulphate (SDS) was added to a final concentration of 0.5% (wt/vol).
4. Add an equal volume of phenol equilibrated with 0.02 M sodium acetate (pH 5.2) and
incubate the mixture at 65°C for 5 minutes with gentle shaking.
5. Again centrifuge the mixture at 12,000X g at room temperature for 5 minutes.
6. Extract the aqueous phase with an equal volume of phenol-chloroform (1:1, vol/vol),
centrifuge as above, and transfer to a sterile 30-mL Corex centrifuge tube.
7. Add three volumes of 100% ethanol, and incubate the mixture at – 80°C for 30 minutes.
8. Collect the RNA precipitate by centrifugation at 12,000X g at 4°C for 30 minutes.
9. Dissolve the RNA pellet in 300 mL of TE buffer and check on an agarose gel
(Figure 3.1).
4981
3638
(Lane 1: The molecular sizes
1908 of RNA markers;
955 Lane 2: RNA isolated from
623 flock house virus).
281
1 2
Figure 3.1 Agarose gel electrophoresis of RNA.
Buffers
(i) 0.02 M Sodium acetate
2.722 g in 1000 mL distilled water
(ii) 1 mM EDTA
0.372 g in 1000 mL distilled water
3.2 ISOLATION OF mRNA FROM CULTURED MAMMALIAN CELLS

Protocol
1. Remove the medium from the cells and wash the monolayer three or four times with ice-
cold phosphate buffered saline (PBS) and 2 mL of ice-cold PBS to each of the plate and
allow standing them on a bed of ice.
2. Scraps off the cell sheet each plate in-turn by using a rubber policeman with a wide-
mouthed pipette, transfer the cell suspension into a corex centrifuge tube and store on
ice and remove all the cells from all the plate.
3. Centrifuge at 200X g for 5 minutes at 4°C.
4. Remove the supernatant by aspiration and resuspend the cell pellet in ice-cold lysis buffer
(0.25 mL/85 mm dish).
5. Vortex for 10 seconds and then underlay the cell suspension with an equal volume of
lysis buffer containing sucrose (24% w/v) and NP-40 (nonyl phenoxylpolyethoxy
lethanol 1%)/CA-630. Store in ice for 5 minutes.
6. Centrifuge at 10,000 rpm for 20 minutes at 4°C in a swing-out rotor.
7. Recover the turbid upper (cytoplasmic) layer and add equal volume of 2X Proteinase K
buffer. Add Proteinase K to a final concentration of 200 mg/mL. Mix and incubate at
37°C for 30 minutes (If nuclear RNA is also to be prepared discard the clear sucrose
phase and resuspend the nuclear pellet at the bottom of the centrifuge tube in lysis buffer
(0.25 mL per 85 mm disk).
8. Add equal volume of 2X Proteinase K buffer to a final concentration of 200 mg/mL.
Disrupt the nuclei and shear the liberated DNA by repeatedly squirting the viscous
solution through a sterile 19-gauge hypodermic needle. Incubate at 37°C for 30 minutes.
9. Remove the proteins by extracting once with phenol/chloroform.
10. Recover the aqueous phase, add 2.5 volume of ethanol and store at –20°C for at least
2 hours.
11. Centrifuge for 10 minutes at 500X g at 0°C. Discard the supernatant and wash the pellet
with 75% ethanol containing 0.1 M sodium acetate (pH 5.2).
12. Redissolve nucleic acids in a small volume (50 mL/85 mm dish) of 50 mM Tris-Cl
(pH 7.5) and 1 mM EDTA.
13. Then add MgCl2 to 10 mM. Add pancreatic DNase I from which RNase has been
removed either by chromatography or agarose-5¢-(4-amino phenyl phosphoryl) uridine
2¢ (3¢) phosphate. Incubate for 30 minutes at 37°C.
14. Add EDTA and SDS to a final concentration of 10 mM and 0.2% respectively.
15. Extract the solution once with phenol/chloroform.
16. Add sodium acetate (pH 5.2) to 0.3 M and mix well and precipitate the nucleic acids with
2 volumes of ice cold ethanol. Wash the RNA in 70% ethanol.
17. Store the RNA in –70°C after dissolving in TE buffer.
18. Cytoplasmic RNA may be further purified and free of any contaminating Oligodeoxy-
ribonucleotides by chromatography on Oligo (dT) - cellulose by the following method.
19. Resuspend the pellet in 20% sodium acetate by repeated pipetting up and down.
Isolation of RNA from Bacteria and Cultured Mammalian Cells 25
20. Centrifuge for 10 minutes in an Eppendorf centrifuge tube.

21. Discard the supernatant and redissolve the pellet in TE buffer. Precipitate with ethanol
(Cytoplasmic RNA can usually be purified by oligo (dT) chromatography without DNase
treatment).
Buffers
(i) Lysis buffer
NaCl – 0.14 M
MgCl2 – 1.5 mM
Tris-Cl (pH 8.6) – 10 mM
NP-40 – 0.5%
(ii) 2X Proteinase K buffer
Tris-Cl (pH 7.5) – 0.2 M
EDTA – 25 mM
NaCl – 0.3 M
SDS – 2% w/v
Add Proteinase K to a final concentration of 200 mg/mL mix and incubate at 37°C for
30 minutes.
REFERENCES
Avison, M. (2006), Measuring Gene Expression, 1st ed., Taylor & Francis.
Chirgwin, J.J., A.E. Przbyla, R.J. MacDonald and W.J. Rutter (1979), Isolation of biologically
active ribonucleic acid from sources enriched in ribonuclease, Biochemistry 18, p. 5294.
Han, J.H., C. Stratowa and W.J. Rutter (1987), Isolation of full-length putative rat
lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning,
Biochemistry, 26, pp. 1617–1625.
Laitinen, J. (2002), Direct isolation of poly-A(+) mRNA from tissue culture cells,
Technical Note Molecular Biology, pp. 1–2.
Schneemann, A. and D. Marshall (1998), Specific encapsidation of nodavirus RNAs
is mediated through the C terminus of capsid precursor protein alpha, J. Virol, 72,
pp. 8738–8746.
Ullrich, A., J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W.J. Rutter and H.M. Goodman (1977),
Rat insulin genes: Construction of plasmids containing the coding sequences, Science, 196,
p. 1313.
4
Estimation of Nucleic Acids
Nucleic acids are present in all living cells and are responsible for storage and transmission of
genetic information. Chemically, nucleic acids are polymers of nucleotides. The nucleotides
contain three components, viz., a nitrogenous base (either purine or a pyrimidine), a pentose
sugar (either deoxyribose in the case of DNAs or ribose in the case of RNAs) and a phosphate
group connected to the sugar. The nucleotides are covalently connected to the phosphate groups
by phosphodiester bonds. Estimation of nucleic acids is important almost in each step of
recombinant DNA and genetic engineering experiments. Nucleic acids absorb UV very strongly
in the UV range at ~260 nm. The amount of UV absorbed is directly proportional to the quantity
of DNA present in the solution. Therefore, they can be quickly and conveniently estimated by
spectrophotometric methods. However, other methods such as colorimetric methods are also
used to estimate DNAs and RNAs in crude preparations.
4.1 ESTIMATION OF DNA

4.1.1 UV Quantitation of DNA by UV Absorbance Spectrophotometry
DNA absorbs ultraviolet light due to its highly conjugated nature. DNA may thus be easily
quantitated in a UV spectrophotometer. Typically, 1 OD260 (i.e. a solution having an absorbance
of one unit at 260 nm with a path length of 1 cm) is said to correspond to a concentration of
30–37 mg per mL. This is for single stranded DNA with an equal mixture of each of the four
bases. RNA or double stranded DNA has values of 40 and 50 mg per mL, respectively.
For work with DNA longer than probes and primers, these assumptions are valid because
the constituent bases are usually fairly evenly represented. With oligos, however, the base
composition may be highly skewed. For instance, 1 OD of a sequence of all cytidines would
correspond to 39.4 mg/mL and 1 OD of a sequence of all adenosine would correspond to
22.8 mg/mL. Since the composition and sequence of the oligo are usually known, this information
may be used to calculate individualized values for more accurate quantitation.
Protocol
1. Switch on power with UV spectrophotometer
2. Switch on UV light
26
Estimation of Nucleic Acids %
3. Set wavelength at 260 nm

4. Preheat for 10 seconds
5. Keep blank distill water in the cuvettes
6. Set zero
7. Add 2 mL DNA/RNA solutions in one cuvette
8. Measure the OD 1 OD is equal to 50 mg/mL for double stranded DNA and 40 mg/mL
for single stranded DNA/RNA and 20 mg/mL for oligonucleotides.
9. The ratio OD at 260 nm/OD at 280 nm should be 1.8 for DNA and 2 for RNA.
10. Then the DNA/RNA preparation is pure.
If there is contamination with protein or phenol, the OD/OD280 will be significantly less
than the values given above, and the accurate quantitation of the amount of nucleic acid
will not be possible.
4.1.2 TD-20/20 Luminometer Method for DNA Quantitation

The Turner BioSystems TD-20/20 Luminometer, in combination with Promega’s new DNA
quantitation system, provides a precise and sensitive method for the detection of linear double
stranded DNA (dsDNA) including PCR fragments. Plasmid and chromosomal DNA can be
quantitated following linearization. The measurement is based upon a series of coupled enzymatic
reactions that produce a light signal, proportional to the amount of linear DNA in a sample. The
DNA quantitation system is more precise than spectrophotometric or agarose gel analyses and
is able to detect picogram amounts of DNA. The assay should be used in the linear range of
20 pg to 1 ng of total DNA added per reaction and can quantitate linear dsDNA species up to
6,000 bp in size. Estimation of DNA amount is dependent upon mass and not the number of DNA
ends or nicks in the DNA for fragments < 6,000 bp.
Since, the DNA quantitation method depends upon the production of ATP by enzymatic
reactions; extra care must be taken to ensure that the samples do not contain dNTPs or NTPs,
which can be used to form ATP in the reactions. Unless a DNA sample is contaminant-free, a
control not containing T4 DNA polymerase should be performed. This control reaction will give
the contribution of signal from materials other than dsDNA. Furthermore, the sample should not
contain any ATPase activity.
Materials required
∑ TD-20/20 luminometer (P/N 2020-000)
∑ 8 mm test tube adapter (P/N 2020-314)
∑ 8 mm ¥ 50 mm test tubes (P/N 20-015)
∑ Microfuge tubes (Sterile)
∑ Micropipettes (aerosol resistant tips are recommended)
∑ Sterile 10 mM Tris-HCl (pH 7.3–7.5)
∑ Sterile TE buffer (pH 7.3–7.5)
∑ Sterile deionized water
∑ DNA quantitation system containing:
∑ 2 ¥ 1 mL, DNA quantitation buffer solution containing ADP
∑ 2 ¥ 500 u, T4 DNA polymerase (5–10 u/mL)

∑ 50 mL, sodium pyrophosphate (40 mM)
∑ 15 mL, Nucleoside diphosphate kinase (NDPK) enzyme solution
∑ 200 mL, DNA quantitation DNA standard (5 ng/mL ± 5%)
∑ 1 vial, ENLITEN® Luciferase/Luciferin reagent
∑ 1 vial, ENLITEN® Reconstitution buffer (12 mL).
Protocol
ENLITEN luciferase/luciferin reagent preparation: Lightly tap the vial of ENLITEN L/L
reagent (supplied by Promega) before opening to gather all of the dried material to the bottom
of the vial. Transfer 12 mL of the ENLITEN L/L reconstitution buffer into the vial of ENLITEN
L/L reagent. Replace the stopper carefully, and gently invert the vial several times to dissolve the
contents. Do not shake the dissolved ENLITEN® L/L reagent. Equilibrate the reconstituted
ENLITEN L/L reagent to room temperature for a minimum of 30 minutes and a maximum of
8 hours. Unused reconstituted reagent should be dispensed into sterile microcentrifuge tubes and
frozen at –20°C.
Reaction master mix preparation: Thaw the enzyme and buffer solution and keep on ice.
Mix the reagents well by gentle inversion or vortexing. Prepare the reaction master mix just prior
to use. Determine the number of reactions to be performed for samples and for preparing a
standard curve. All samples and controls should be performed in duplicate or triplicate. Prepare
a master mix using Table 4.1 as a guide. Add the components in the order listed to a fresh
microcentrifuge tube, mix gently and keep the solution on ice.
TABLE 4.1 Volume of master mix components required by number of reactions

3
Component 1–9 10 Experiment
DNA quantitation n ¥ 15.5 mL n ¥ 15.5 mL _ ¥ __ mL = ___ mL
buffer solution
Sodium pyrophosphate n ¥ 0.5 mL n ¥ 0.5 mL _ ¥ __ mL = ___ mL
NDPK enzyme solution n ¥ 1.0 mL n ¥ 0.1 mL _ ¥ __ mL = ___ mL
Water, sterile deionized — n ¥ 0.9 mL _ ¥ __ mL = ___ mL
T4 DNA polymerase n ¥ 1.0 mL n ¥ 1.0 mL _ ¥ __ mL = ___ mL
Total volume n ¥ 18.0 mL n ¥ 18.0 mL _ ¥ __ mL = ___ mL
Instrument set-up: Turn on instrument (luminometer) and allow to warm up for at least
5 minutes (600 seconds).
Adjust settings so that:
Delay period = 3 seconds
Integrate period = 15 seconds
Replicates = 1
Protocol
1. Prepare the reconstituted ENLITEN L/L reagent. Allow to equilibrate to room temperature.
2. Prepare samples for the standard curve by making dilutions of the provided DNA
quantitation DNA standard. The DNA standard should be mixed well. Dilutions can be
Estimation of Nucleic Acids '
made using 10 mM Tris-HCl (pH 7.3–7.5) or TE buffer (10 mM Tris, 1 mM EDTA

[pH 7.3–7.5]). The prepared samples should span the region of interest in the linear
range of the curve.
3. If needed, dilute the DNA samples to be tested to 10–500 pg/mL using 10 mM Tris-HCl
(pH 7.3–7.5) or TE buffer.
4. Prepare the reaction master mix.
5. Aliquot 18 mL of master mix into each prelabelled microcentrifuge tube.
6. Add 2 mL of DNA solution (from duplicate or triplicate reactions) to each of the tubes
and vortex gently. Include a negative control containing 2 mL of the buffer used to dilute
the standard.
7. Incubate at 37°C (in a heating block or water bath) for 8–10 minutes. Do not incubate
longer.
8. Place samples on ice and analyze as soon as possible.
9. Remove 15 mL from one reaction mix and place into 100 mL of reconstituted ENLITEN®
L/L reagent. Mix gently.
10. Immediately read the sample by pressing GO on the TD-20/20. Repeat Steps 9 and 10
until all samples are read.
DNA concentration calculation
1. Calculate the average of duplicate or triplicate sample values.
2. Subtract the averaged value for the ‘no DNA’ blank reaction from that obtained for the
other samples. This yields net light unit (LU).
3. Net average LU (y) = Average LU sample – Average LU No DNA Control
4. Generate the standard curve by plotting DNA concentration vs. net light units and
drawing a best-fit line (of the form y = mx, or y = m x + b).
5. Determine the concentration of the unknown samples by comparing the net light units
seen for these samples against those seen for the standard curve (Figure 4.1).
DNA Quant standard curve
700
600
500
RLU values
400
300
200
100
0
0 50 100 150 200 250 300
DNA concentration (pg/mL)
Figure 4.1 DNA quantification graph.
4.1.3 Diphenylamine Method

This method can be used on relatively crude extracts where direct measurements of DNA by
ultraviolet absorbency are not possible. The assay is specific for deoxyribose, although very high
concentrations of ribose or sucrose must be avoided.
! Laboratory Manual for Genetic Engineering
Principle
In the presence of strong acid, the deoxyribose moieties of DNA form hydroxylevulinic acid. The
hydroxylevulinic acid reacts with diphenylamine and produces a blue coloured complex. The
colour is measured at 595 nm or using a red filter. It is important to note that only the deoxy-
moieties of purine nucleotides (adenine and guanine) react and produce the colour.
Protocol
1. Pipette 0.05 mL to 1.0 mL of DNA stock solution into clean tubes and make the volume
to 1.0 mL with distilled water. Keep a blank.
2. Add 5 mL of diphenylamine reagent to each tube and vortex. Cover the tubes with
aluminium foil and secure it with rubber bands.
3. Now place the tubes in a boiling water bath for 10 minutes.
4. Cool the tubes to room temperature and read the absorbancy at 595 nm.
5. Draw a standard graph using the values obtained for A 595 as function of DNA
concentration.
6. Vortex and place the tubes containing DNA samples of unknown quantity in boiling water
bath for 10 minutes, and cool to room temperature and read the absorbance at 595 nm.
7. Calculate the quantity of DNA of unknown samples from the standard graph.
Note
A diphenylamine reagent is not water soluble. Therefore, rinse the cuvettes, glasswares and test
tubes in ethanol before washing them in water.
Buffer
(i) Preparation of diphenylamine (DPA) reagent: Dissolve 1.0 g diphenylamine
(AR grade) in 97.5 mL of glacial acetic acid and then add 2.5 mL of concentrated
sulphuric acid. Mix well and store the reagent in a brown bottle at room temperature (this
reagent is stable for months).
DNA stock solution

Dissolve calf thymus DNA or equivalent type at 0.2 mg/mL in 5 mM NaOH. Store at 4°C (this
reagent is stable for at least 6 months). Highly polymerized DNAs should be dissolved overnight
at 4°C on a shaker.
4.2 ESTIMATION OF RNA

4.2.1 Orcinol Method
The method can be used on relatively crude preparations of RNA where direct measurements of
RNA by ultraviolet absorbance are not possible.
Principle
Ribose moieties present in ribonucleic acids react with hydrochloric acid and produce furfural.
The furfural thus formed is reacted with orcinol in the presence of ferric ions to give a brilliant
Estimation of Nucleic Acids !
green colour. The intensity of the colour is measured at 665 nm (the purine nucleotides are
generally more reactive than pyrimidine nucleotides).
Protocol
1. Pipette out 0.05 mL to 2.0 mL of RNA stock solution into clean test tubes and make up
the final volume to 2.0 mL with distilled water.
2. Add 4.0 mL of the orcinol reagent to each tube and vortex.
3. Cover the tubes with aluminium foil, secure with a rubber band and place the tubes in
a boiling water bath for 15 minutes.
4. Keep the tubes in a tray containing tap water to room temperature and measure the
absorbency at 665 nm.
5. Draw a standard graph by plotting the values as a function of RNA concentration and
calculate the unknown RNA concentration from the standard graph and express the
results as ribose equivalent.
Buffers
(i) 1% Orcinol solution: Dissolve 1.0 g orcinol in 5 mL ethanol and make the volume to
100 mL with distilled water. It can be stored in the refrigerator for several months.
(ii) Concentrated HCl
(iii) 10% Ferric chloride solution (prepare afresh): Dissolve 2 g of FeCl 3 ◊6H2O in distilled
water and make up the volume to 20 mL.
(iv) Preparation of orcinol reagent (prepare afresh): Mix 10 mL of 10% ferric chloride
solution to 390 mL of conc. HCl. Add the mixture slowly into 100 mL of 1% orcinol
solution with stirring. Make up the final volume up to 500 mL.
(v) RNA stock solution: Dissolve yeast RNA or equivalent at 0.1 mg/mL in distilled water
(store at –20°C).
REFERENCES
Hoisington, D., M. Khairallah and D. Gonzalez-de-Leon (1994), Laboratory Protocols: CIMMYT
Applied Biotechnology Center, 2nd ed., Mexico, D.F.: CIMMYT.
Kamali, M. and H. Manhouri (1968), A Modified Orcinol Reaction for RNA Determination, Clinical
Chem., 15(5), pp. 390–392.
! Laboratory Manual for Genetic Engineering
5
Restriction Digestion and Ligation of DNA
Restriction enzyme digestion is performed by incubating double-stranded DNA molecules with

an appropriate amount of restriction enzyme, in its respective buffer as recommended by the
supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride
concentration in the reaction varies for different enzymes, and a set of three-standard buffers
containing three concentrations of sodium chloride are prepared and used when necessary.
Typical digestions include a unit of enzyme per microgram of starting DNA, and one restriction
enzyme unit is usually defined as the amount of enzyme needed to completely digest one
microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions
usually are incubated for 1–3 hours, to ensure complete digestion, at the optimal temperature for
enzyme activity, typically at 37°C.
DNA ligations are performed by incubating DNA fragments with appropriately digested
cloning vector in the presence of ligation buffer containing ATP and T4 DNA ligase. For random
shotgun cloning, sonicated or nebulized fragments, in which the fine mist created by forcing a
DNA solution through a small hole in the nebulizer unit is collected. The size of the fragments
obtained by nebulization is determined chiefly by the speed at which the DNA solution passes
through the hole, altering the pressure of the gas blowing through the nebulizer, the viscosity of
the solution, and the temperature. Nebulization is easy, quick, and requires only small amounts
of DNA (0.5–5 mg). The resulting DNA fragments are distributed over a narrow range of sizes
(700–1330 bp). It requires ligation of DNA before nebulization and end-repair afterward, are
ligated either to Sma I linearized, dephosphorylated double-stranded M13 replicative form or pUC
vector by incubation at 4°C overnight. A practical range of concentrations is determined based
on the amount of initial DNA and several different ligations, each with an amount of insert DNA
within that range, are used to determine the appropriate insert to vector ratio for the ligation
reaction. In addition, several control ligations are performed to test the efficiency of the blunt-
ending process, the ligation reaction, and the quality of the vector. These usually include parallel
ligations in the absence of insert DNA to determine the background clones arising from self-
ligation of inefficiently phosphatased vector. Parallel ligations are also performed with a known
blunt-ended insert or insert library, typically an Alu I digest of a cosmid, to ensure that the blunt-
ended ligation reaction would yield sufficient insert containing clones, independent of the repair
process.
32
Restriction Digestion and Ligation of DNA !!
5.1 RESTRICTION DIGESTION OF DNA

Protocol
1. Prepare the restriction digestion reaction mixture by adding the following reagents in the
order listed to a microcentrifuge tube:
DNA (1 mg) = 10 mL
10X restriction enzyme assay buffer = 2 mL
Restriction enzyme (1–10 units) = 1 mL
Sterile ddH20 = 7 mL
Total restriction volume = 20 m L
Note
(i) If desired, more than one enzyme can be included in the digest if both enzymes are
active in the same buffer and the same incubation temperature.
(ii) The volume of the reaction depends on the amount and size of the DNA being
digested. Larger DNAs should be digested in larger total volumes (between
50–100 mL).
(iii) The supplier’s catalogue should be referred to the chart of enzyme activity in a
range of salt concentrations to choose the appropriate assay buffer (10X High, 10X
Medium, or 10X Low salt buffers, or 10X Sma I buffer for Sma I digestions).
2. Gently mix the restriction digestion reaction mixture by pipetting up and down and
incubate the reaction at the appropriate temperature (typically at 37°C) for 1–3 hours.
3. After incubation for 1–3 hours inactivate the enzyme(s) by heating at 65°C for
10 minutes or by phenol extraction. Prior to use for subsequent dephosphorylation or
ligation, an aliquot of the digestion should be assayed by agarose gel electrophoresis
along with non-digested DNA and a size marker, for conformation of the digestion
(Figure 5.1).
5.2 PURIFICATION OF RESTRICTED DNA FRAGMENTS

Protocol
1. Dilute the restricted DNA to 500 mL with TE buffer.
2. Extract once with equal volume of equilibrated phenol (add equal volume of phenol, mix
and centrifuge at 10,000 rpm for 10 minutes at 4°C. Transfer the upper phase into
another sterile tube).
3. Add equal volume of chloroform: isoamyl alcohol (24:1) and extract as in previous step.
4. Take the upper phase and add sodium acetate (pH 4.6) to a final concentration of 0.3 M.
Mix well and add four volume of ice cold 100% ethanol.
5. Mix well and incubate for 1 hour at –20°C and centrifuge at 10,000 rpm for 10 minutes
at 10°C.
6. Decant the supernatant and wash the DNA pellet with 70% ethanol.
7. Air dry the final DNA pellet and resuspend it in 10 mL of sterile double distilled water
for subsequent ligation reaction or 10 mL of TE buffer for storage.
!" Laboratory Manual for Genetic Engineering
M: 1 mg f X 174/HaeIII Markers
A: Undigested DNA;
B: BpuAm1
C: Sac 1
M A B C
Figure 5.1 Agarose gel electrophoresis of restriction fragments produced by cleavage of Ad2
phage DNA.
Buffer
(i) 3 M Sodium acetate (pH 4.6)
(Dissolve sodium acetate salt in less volume of distilled water and adjust the pH to 4.6
with glacial acetic acid and finally make up the volume).
5.3 DNA LIGATION

Protocol
1. Combine the following reagents in a microcentrifuge tube and incubate overnight at
12–16°C:
Digested insert DNA fragments = 4 mL (50 ng/mL)
Digested cloning vector = 2 mL (10 ng/mL)
(with same restriction enzymes)
10X ligation buffer = 1 mL
T4 DNA ligase = 1 mL (10 U/mL)
Sterile ddH2O = 2 mL
Total volume = 10 mL
2. Include control ligation reactions with no insert DNA and with a known blunt-ended
insert (such as Alu I digested cosmid) as controls.
Restriction Digestion and Ligation of DNA !#
3. Transform the ligated DNA in to E. coli either by competent cell transformation or by

electroporation.
4. Score the efficiency of ligation.
REFERENCES
Ausubel, F.M., et al. (1994–2000), Current Protocols in Molecular Biology, vol. 1, John Wiley
& Sons, Inc., Brooklyn, New York.
Sambrook, J. and D.W. Russel (2001), Molecular Cloning: A Laboratory Manual, 3rd ed., Cold
Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press.
!$ Laboratory Manual for Genetic Engineering
6
Polymerase Chain Reaction and
Randomly Amplified Polymorphic DNA
The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a
desired gene. This is necessary to have enough starting template for sequencing or for gene
cloning. Any sequence of DNA can be amplified if the flanking sequences are known. Based on
the flanking sequence, the primers 1 and 2 must be designed and used for PCR amplification of
the desired gene. PCR amplification is done by the following steps:
The cycling reactions: There are three major steps in a PCR, which are repeated for 30 or
40 cycles. This is done on an automated thermo cycler, which can heat and cool the tubes with
the reaction mixture in a very short time based on the set programme.
Denaturation at 94°C: During the denaturation, the double-stranded DNA melts open to single-
stranded DNA, all enzymatic reactions stop (for example: the extension from a previous cycle).
Annealing at 54°C: Ionic bonds are constantly formed between the single-stranded primer and
the single-stranded template. The more stable bonds last a little bit longer (primers that fit exactly)
and on that little piece of double-stranded DNA (template and primer); the polymerase can attach
and starts copying the template. Once there are a few bases built-in, the ionic bond is so strong
between the template and the primer, that it does not break anymore.
Extension at 72°C: This is the ideal working temperature for the polymerase. The primers,
where there are a few bases built-in, already have a stronger ionic attraction to the template than
the forces breaking these attractions. Primers that are on positions with no exact match get loose
again (because of the higher temperature) and will not give an extension of the fragment.
The bases (complementary to the template) are coupled to the primer on the 3¢ side (the
polymerase adds dNTP’s from 5¢ to 3¢, reading the template from 3¢ to 5¢ side, bases are added
complementary to the template).
6.1 IMPORTANT PARAMETERS IN THE PCR

Template DNA quantity (complexity determines ng) and quality (average length) while people
typically measure DNA quantity in ng, the relevant unit is actually moles, i.e, how many copies
36
Polymerase Chain Reaction and Randomly Amplified Polymorphic DNA !%
of the sequence that will anneal with the primers are present. Thus, the amount of DNA in ng
that needs to be added is a function of its complexity. In theory, a single molecule of DNA can
be used in PCR but normally people use between 1000 and 100,000 molecules for eukaryotic
nuclear DNA.
Example for sorghum (genome size = 760 Mb):
760 Mb = 7.6 ¥ 108 bp ¥ 649 daltons/bp = ~ 5 ¥ 1011 grams/mole
20 ng = 2 ¥ 10 –8 grams/5 ¥ 1011 grams/mole = 1 ¥ 10–18 mole
1 ¥ 10 –18 mole ¥ 6 ¥ 1023 molecules/mole = 6 ¥ 104 molecules
Example for a 5 kb plasmid clone:
5 kb = 5 ¥ 103 bp ¥ 649 daltons/bp = 3.2 ¥ 106 grams/mole
1 pg = 1 ¥ 10 –12 grams/3.2 ¥ 106 grams/mole = 3.2 ¥ 10 –18 mole
3.2 ¥ 10–18 mole ¥ 6 ¥ 1023 molecules/mole = 2 ¥ 106 molecules
6.1.1 Tm of Primers
The melting temperature (Tm) of primers should be similar and should be as high as possible, in
order to increase specificity. Tm of the primer to be higher than the reaction temperature of Taq
polymerase (72°C). In practice Tm around 66°C is usually possible with a primer length of
22 or 23 bases. These primers can frequently be used in 2-step PCR.
To calculate Tm for duplex DNA < 50 bp:
Add 2°C for each A or T
Add 4°C for each G or C
The annealing temperature used in PCR should be a function of the Tm of primers and it
should not be much lower unless designed, the primer from heterologous sequence, in which
case one may want to do a gradient PCR.
6.1.2 Mg Concentration
Standard Mg++ concentration is 2 mM, but sometimes the concentration needs to be raised (rarely
lowered) to get a PCR to work. Raising Mg2+ lowers specificity, and is roughly comparable to
lowering the annealing temperature. It may cause multiple bands to appear (or, occasionally,
disappear).
6.1.3 Length of Expected Product

The length of the extension step (72°C) should be a function of the length of the product one
is trying to amplify. A general guideline is 1 minute/kb of product length, but in fact, this is more
than what is needed, particularly if one is doing 3-step (i.e., conventional) PCR, as extension will
take place during the annealing step and during the ramp time. Taq polymerase is a very fast and
very processive enzyme.
!& Laboratory Manual for Genetic Engineering
6.2 POLYMERASE CHAIN REACTION (PCR)

Materials/equipment needed: The following materials/equipment are needed for Polymerase
Chain Reaction (PCR):
∑ Thermocycler
∑ Microcentrifuge (optional)
∑ Micropipette
∑ Micropipette tips
∑ Master mix
For each reaction:
To a 0.2 mL PCR microfuge tube add the following contents:
1. 10 mL of 2.5X master mix which contains:
∑ Sterile water
∑ MgCl2
∑ PCR buffer
∑ Nucleotides (dNTPs)
∑ Taq DNA polymerase
2. 5 mL extracted DNA (DNA template)
3. 5 mL of the forward primer
4. 5 mL of the reverse primer
Protocol
1. Using the micropipet with a clean tip, add 5 mL extracted DNA to the appropriate tube.
2. After adding the DNA, add 10 mL master mix and 5 mL of each of the primers to the
tubes. If necessary, gently tap tube on the counter to get all of the liquid to the bottom
of the tube.
3. Place the tubes into the slots of thermocycler and run for 30–40 cycles with the
following temperature cycle:
∑ Denaturation – 94°C for 30 seconds
∑ Primer annealing – 55°C for 45 seconds
∑ Chain and extension – 72°C for 1.5 minutes
After the cycles are complete, PCR reactions can be refrigerated and can be analysed by
agarose gel electrophoresis. A typical pattern of PCR products are shown in Figure 6.1.
Figure 6.1 Ethidium bromide-stained PCR products after gel electrophoresis.

Polymerase Chain Reaction and Randomly Amplified Polymorphic DNA !'
6.3 RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)

A widely applied approach to characterize eukaryotic DNA and mapping is to use PCR with a
short oligonucleotide primer of arbitary (random) sequence to generate markers. Original PCR
technique has been modified to use random primer giving randomly amplified polymorphic DNAs
(RAPDs). Here, instead of using a pair of oligonucleotide as primers, a single short
oligonucleotide primer could be successfully used. This is the basis of the random amplified
polymorphic DNA method and this RAPD method is adopted most widely for genome mapping
and strain identification.
The RAPD method is based on the probability that given DNA sequences in a genome are
potential templates (complementary to that of the primer) on opposite strands of DNA in opposite
orientation, within a reasonable distance amplifiable by PCR. For most plant materials-primers
that are 9–10 long (random nucleotide sequences with at least 50% G and C are lacking inverted
repeats), are designed to generate on an average 2–10 amplified products. Polymorphism results
mainly due to changes, i.e. presence or absence in the primer binding sites. Amplification products
represent one allele per locus. The RAPD marker could be used for preparation of genetic maps
for a variety of crop plants and thus helpful in crop improvement programmes. The RAPD
method makes use of agarose gels to analyse the PCR products. This technique is used to identify
variation within cultivars, varieties, etc.
The RAPD methods have several more polymorphisms if combined with restriction
digestion, e.g., in wheat- with little genetic variation, digestion of genomic DNA from wheat with
restriction enzymes before RAPD analysis has revealed more polymorphisms.
In order to get reliable and reproducible RAPD results one has to follow a consistent
approach in isolating template DNA, using standard primer, same magnesium ion concentration,
temperature cycling and DNA polymerase for RAPD reaction.
Protocol
1. Prepare a DNA template using CTAB or any method and dilute to10 ng/mL.
2. Add the following to a tube suitable for use in PCR:
(i) 12.5 mL dH2O.
(ii) 2.5 mL dNTPs (from stock solution 1 mM to give a final concentration of 100 mM
for each dNTP).
(iii) 2 mL MgCl2 (25 mM to give a final concentration 2 mM).
(iv) 2.5 mL of reaction buffer (100 mM Tris-HCl, 500 mM KCl at pH 8.3 to give a final
concentration of 10 mM Tris-HCl and 50 mM HCl).
(v) 0.5 mL thermostable DNA polymerase (Taq DNA polymerase) to a final concentra-
tion of 0.02 U/mL.
(vi) 2.5 mL primer (2 mM to give a final concentration of 1 ng/mL).
3. Centrifuge briefly to mix the contents of the tube.
4. Add mineral oil (if necessary for thermal cycler).
5. Cycle at 94°C for 1 minute, 63°C for 1 minute and 72°C for 2 minutes for 45 cycles.
6. Analyse by electrophoresis on agarose gels and detect by ethidium bromide
staining (Figure 6.2).
Figure 6.2 Ethidium bromide-stained RAPD products after gel electrophoresis.
REFERENCES
Sambrook, J. and D.W. Russel (2001), Molecular Cloning: A Laboratory Manual,
3rd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, Ch. 8: In vitro
Amplification of DNA by the Polymerase Chain Reaction.
Williams, J.G.K., Anne R. Kubelik, Kenneth J. Livak, J. Antoni Rafalski and Scott V. Tingey
(1990), DNA polymorphisms amplified by arbitrary primers are useful as genetic markers,
Nucleic Acids Research, 18 (22), pp. 6531–6535.
Electrophoresis of Nucleic Acids "
7
Electrophoresis of Nucleic Acids
The term, gel in this instance, refers to the matrix used to contain and then separate the target
molecules. In most cases the gel is a cross-linked polymer whose composition and porosity is
chosen, based on the specific weight and composition of the target to be analyzed. When
separating proteins or small nucleic acids (DNA, RNA or oligonucleotides), the gel is usually
composed of different concentrations acrylamide and cross-linker, producing different sized
mesh networks of ployacrylamide. When separating larger nucleic acids (greater than a few
hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet
porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled
using appropriate safety precautions to avoid poisoning.
Electrophoresis refers to the electromotive force (EMF) that is used to move the molecules
through the gel matrix. By placing the molecules in wells in the gel and applying an electric
current, the molecules will move through the matrix at different rates, usually determined by
mass, towards the positive anode if negatively charged or towards the negative cathode, if
positively charged.
The agarose gel electrophoresis is a method used in biochemistry and molecular biology to
separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic
acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter
molecules move faster and migrate farther than longer ones.
7.1 AGAROSE GEL ELECTROPHORESIS OF DNA

Agarose is a linear polymer composed of alternating residues of D- and L-galactose joined by
a-(1 Æ 3) and b-(1 Æ 4) glycosidic linkages. The L-galactose residue has an anhydro bridge
between third and sixth positions. Chains of agarose from helical fibres that aggregate into
supercoiled structures with a radius of 20–30 nm. Gelling of agarose results in a three-
dimensional mesh of channels whose diameters range from 50 nm to > 200 nm.
Commercial agarose polymers contain ~800 galactose residues per chain. However, agarose
is not homogenous: The average length of polysaccharide chains varies from batch to batch and
from manufacturer to manufacturer. In addition, lower grades of agarose may be contaminated
41
with other polysaccharide, as well as salts and proteins. This variability can affect the gelling/
melting temperature of agarose solutions, the sieving of DNA and the ability of DNA recovered
from the gel to serve as a substrate in enzymatic reactions. These potential problems can be
minimized by using special grades of agarose that are screened for the presence of inhibitors and
nucleases and for minimal background fluorescence after staining with ethidium bromide.
7.1.1 The Rate of Migration of DNA through Agarose Gels

The following factors determine the rate of migration of DNA through agarose gels:
The molecular size of the DNA

Molecules of double-stranded DNA migrate through gel matrices at rates that are inversely
proportional to the log10 of the number of base pairs. Larger molecules migrate more slowly
because of greater frictional drag and because they move through the pores of the gel less
efficiently than the smaller molecules.
The concentration of agarose

A linear DNA fragment of a given size migrates at different rates through gels containing different
concentration of agarose. There is a linear relationship between the logarithm of the electrophoretic
mobility of the DNA (m) and the gel concentration (i) that is described by the equation:
log m = log mo – K r i
where mo is the free electrophoretic mobility of DNA and K r is the retardation coefficient, a
constant related to the properties of the gel and the size and shape of the migrating molecules.
The conformation of the DNA

Superhelical circular (form I), nicked circular (form II) and linear (form III) DNAs migrate
through agarose gels at different rates. The relative mobilities of three forms depend primarily
on the concentration and type of agarose used to make the gel, but they are also influenced by
the strength of the applied current, the ionic strength of the buffer and the density of superhelical
twists in the form I DNA. Under some conditions, form I DNA migrates faster than form III
DNA; under other conditions, the order is reversed. In most cases, the best way to distinguish
between the different conformational forms of DNA is, simplify to include in the gel a sample
of untreated circular DNA and a sample of the same DNA that has been linearized by digestion
with a restriction enzyme that cleaves the DNA in only one place.
The presence of ethidium bromide in the gel and electrophoresis buffer

Intercalation of ethidium bromide causes a decrease in the negative charge of the double-stranded
DNA and an increase in both its stiffness and length. The rate of migration of the linear DNA-
dye complex through gels is consequently retarded by a factor of ~15%.
The applied voltage

At low voltages, the rate of migration of linear DNA fragments is proportional to the voltage
applied. However, as the strength of the electric field is raised, the mobility of high-molecular-
weight fragments increases differentially. Thus, the effective range of separation in agarose gels
Electrophoresis of Nucleic Acids "!
decreases with increased voltage. To obtain maximum resolution of DNA fragments > 2 kb in
size, agarose gels should be run at no more than 5–8 V/cm.
The type of agarose

The two major classes of agarose are standard agarose and low melting temperature agarose. A
third and growing class consists of intermediate melting/gelling temperature agarose, exhibiting
properties of both the above two major classes.
The electrophoresis buffer

The electrophoretic mobility of DNA is also affected by the composition and ionic strength of
the electrophoresis buffer. In the absence of ions (e.g., if water is substituted for electrophoresis
buffer in the gel or in the reservoirs), electrical conductivity is minimal and DNA migrates slowly,
if at all. In buffer of high ionic strength (e.g., if 10X electrophoresis buffer is mistakenly used),
electrical conductance is very efficient and significant amount of heat is generated resulting the
melting of gel and denaturing the DNA molecules. For separating DNA molecules of desired sizes
on agarose gels, one has to take all these above factors into consideration for getting the optimum
resolution of DNA.
Protocol
A. Casting the gel
1. Make 25 mL of a 0.7% (w/v) solution of agarose in 1X TBE or in 1X TAE buffer
(Tables 7.1 and 7.2).
2. Weigh the container with the mixture and record the mass.
3. Heat the mixture to boiling point, using the microwave oven. Examine the flask and
continue boiling if any agarose is undissolved.
4. Weigh the container with the mixture again and add deionized water to compensate for
loss of mass during boiling.
5. Allow the agarose to cool for 3–5 minutes at room temperature before pouring the gel.
6. Make sure the wedges are in place firmly against the ends of the gel casting tray. Pour
all of the agarose solution into the gel casting tray, being careful not to overflow the tray.
Place the comb in the proper place and leave the gel to cool and solidify.
TABLE 7.1 Buffers for agarose gel

Buffer Working concentration Stock concentration/litre
Tris-acetate (TAE) 0.04 M Tris-acetate 50X: 242 g Trizma base
0.001 M EDTA 57.1 mL Glacial acetic acid
100 mL 0.5 M EDTA (pH 8.0)
Tris-phosphate (TPE) 0.08 M Tris phosphate 10X: 108 g Trizma base
0.002 M EDTA 15.5 mL of 85% Phosphoric acid
(1.679 mg/mL)
40 mL of 0.5 M EDTA (pH 8.0)
Tris-base (TBE) 0.089 M Trizma base 5X: 54 g Trizma base
0.089 M Boric acid 27.5 g Boric acid
0.002 M EDTA 20 mL of 0.5 M EDTA (pH 8.0)
"" Laboratory Manual for Genetic Engineering
TABLE 7.2 Agarose percentage and separation range of linear DNA molecules
Amount of agarose in gel% Efficient range of separation of
linear DNA molecules (kb)
0.3 5–60
0.6 1–20
0.7 0.8–10
0.9 0.5–7
1.2 0.4–6
1.5 0.2–4
2.0 0.1–3
B. Preparing the samples

1. While the gel is cooling, prepare the DNA samples by adding 1 mL of (6X) tracking dye
to 5 mL of DNA preparation. The tracking dye is bromophenol blue in a 50% glycerol
solution. Adding tracking dye to the sample will increase its density so it falls into the
well of the gel and provides a visible marker to monitor the progress of electrophoresis.
Also prepare a molecular size standard by mixing 5 mL of the 1 kb ladder with 1 mL of
tracking dye (Table 7.3).
TABLE 7.3 DNA gel loading dyes

Buffer type 6X buffer Storage temperature
I 0.25% Bromophenol blue 4°C
0.25% Xylene cyanol
40% (w/v) Sucrose in water
II 0.25% Bromophenol blue R.T
0.25% Xylene cyanol
15% Ficoll (type 400) in H2O
III 0.25% Bromophenol blue 4°C
0.25% Xylene cyanol
30% Glycerol in water
IV 0.25% Bromophenol blue 4°C
40% (w/v) Sucrose in water
C. Loading and running the gel

1. Remove the wedges from the casting tray and fill the buffer reservoir with 1X TBE or
with 1X TAE buffer until the buffer is 1–2 mm deep over the gel.
2. Carefully remove the comb by lifting it straight out of the gel.
3. Carefully pipette each mixture (DNA mixed with tracking dye) (6 mL) into a well in the
gel. Load one well with the prepared 1 kb ladder.
4. After all the lanes have been loaded, connect the leads from the power supply to the gel
running tank. Make sure the gel is oriented correctly [wells at negative (black) end, DNA
will “run to the red” positive end]. Set the output level to 100 volts and turn the power on.
5. Run the gel until the tracking dye is approximately 3/4 the way across the gel.
Electrophoresis of Nucleic Acids "#
D. Staining the DNA in the gel with ethidium bromide

1. After turning the power off, remove the gel from the gel tank and submerge it in the
ethidium bromide staining solution (0.5 mg/mL) in distilled water. Allow the gel to stain
for 5 minutes.
2. Visualize the DNA bands under UV trans-illuminator with protective goggles (Figure 7.1).
The first lane

contains a DNA
10,000 bp ladder for sizing,
and the other
four lanes show
3,000 bp variously-sized
DNA fragments.
500 bp
Figure 7.1 Agarose gel electrophoresis of DNA samples.
Buffers
(i) TBE buffer (10X) 1000 mL
Tris base – 0.89 M (107.81 g/L)
EDTA (disodium) – 0.02 M (7.44 g/L)
Boric acid – 0.89 M (55.0 g/L)
Autoclave for 20 minutes
(ii) 6X loading dye
Tris-HCl (pH 7.6) – 10 mM
Bromophenol blue – 0.03%
Xylene cyanol – 0.03%
Glycerol – 60%
EDTA – 60 mM
(iii) Ethidium bromide 10 mg/mL stock (working conc. 0.5 mg/mL)
"$ Laboratory Manual for Genetic Engineering
7.2 POLYACRYLAMIDE GEL ELECTROPHORESIS OF DNA

Polyacrylamide gels are used to analyse fragments of DNA less than 1 kb in length. They may
be cast in a variety of polyacrylamide concentration ranging from 3.5% to 20%, depending on
the sizes of the fragments of interest. Polyacrylamide gels are poured between two glass plates
that are held apart by spacers. They are invariably run in the vertical position.
Protocol
1. Calculate the volume of acrylamide solution required and prepare solution polyacrylamide
with TBE (Table 7.4).
TABLE 7.4 Gel recipes

Reagents Polyacrylamide gel (%)
3.5 5.0 8.0 12.0 20.0
30% acrylamide 11.6 16.6 26.6 40.0 66.6
Distilled water 76.3 71.3 61.3 47.9 21.3
3% Ammonium persulphate 2.1 2.1 2.1 2.1 2.1
10X TBE 10.0 10.0 10.0 10.0 10.0
Total volume in mL 100.0 100.0 100.0 100.0 100.0
2. Prepare the required quantity of solution in a side arm flask and deaerate it completely.
3. Prepare the glass plates for pouring the gel. Wash the plate with warm detergent solution
and rinse well in tap water and then deionized water.
4. Lay the inner plate in position, resting on the spacer base. Bind the entire length of the
two sides and the bottom of the plates with Whatman 3 mm tap (electrical) to make a
watertight seal.
5. Add 30 mL of TEMED to each 100 mL of deaerated acrylamide mix and pour immedi-
ately into the space between the two plates without any air bubbles and avoid any leakage.
6. Fill almost to the top. Keep the remaining acrylamide solution at 4°C to reduce the speed
of polymerization.
7. Insert appropriate comb immediately, being careful not to allow any air bubbles trapped,
under the teeth. The top of teeth should be slightly higher than the top of the glass plate.
8. Allow the polyacrylamide to polymerize at room temperature for 60 minutes, adding
additional acrylamide if it gets retracted significantly.
9. After polymerization, remove the comb and immediately rinse out the wells with water.
Remove the electrical tap from the sides of the gel.
10. Attach the gel to the electrophoresis tank with large bull clips for the sides and shoulders.
11. Fill the reservoir with 1X TBE buffer.
12. Use a pasteur pipette to flush out the wells with electrophoresis buffer. If this is not done,
diffused waxy bands of DNA will be observed.
13. Load samples of DNA using a micro-pipette. The capacity of polyacrylamide gel is quite
high and up to 1 mg DNA per band can be loaded into a slot of 0.5 cm long and 0.2 cm
wide.
Electrophoresis of Nucleic Acids "%
14. Gel runs at voltage gradients between 1 V/cm and 8 V/cm. At higher voltages, heating
of the gel may cause lowering of DNA bands or even melting of strands of small DNA
fragments. The marker dye migrates in polyacrylamide gel in 1X TBE buffer at the same
rate as DNA fragments of the following sizes (Tables 7.5 and 7.6).
TABLE 7.5 Concentration of acrylamide and sizes of nucleotides

Acrylamide (%) Effective range of nucleotides
3.5 100–1000
5.0 80–500
8.0 60–400
12.0 40–200
20.0 10–100
TABLE 7.6 Migration of marker dyes in polyacrylamide gels

% Gel BPB Xylene cyanol
3.5 100 460
5.0 65 260
8.0 45 160
12.0 20 70
20.0 12 45
The numbers are the appropriate sizes of fragments of DNA (in nucleotides
pairs) with which the dyes would co-migrate).
15. After electrophoresis, the gel can be stained with 0.5 mg/mL of ethidium bromide in
1X TBE buffer and the DNA bands can be visualized under UV trans-illuminator and
photographed.
Buffers
(i) Stock solution (100 mL)
Acrylamide – 30% gel
Acrylamide – 29.0 g
N, N’methylene bisacrylamide – 1.0 g
Distilled water up to 100 mL
(ii) TBE buffer as in agarose gel
(iii) 3% Ammonium persulphate 10 mL
Ammonium persulphate – 0.3 g
(Prepare fresh solution daily)
"& Laboratory Manual for Genetic Engineering
7.3 ELECTROPHORESIS OF RNA THROUGH GELS

CONTAINING FORMALDEHYDE
Protocol
1. Agarose is prepared by melting 1% agarose in distilled water, cooling to approximately
60°C (hand-bearing temperature) and adding 40% formaldehyde and 10X MOPS
(morpholinopropanesulphonic acid) to give 2.2 M formaldehyde and 1X MOPS,
respectively. Example: For 50 mL of a 1% agarose gel, melt 0.5 g agarose in 37 mL H2O,
cool to hand bearing temperature, add 5 mL 10X MOPS buffer and 8.75 mL 40%
formaldehyde.
2. Electrophoresis buffer is prepared in 1X MOPS, 2.2 M formaldehyde.
3. RNA samples are prepared by adding up to 25 ng of RNA in a maximum of 5 mL sterile
H2O, to 15 mL RNA denaturation buffer.
4. 1 mL 10 mg/mL ethidium bromide is added to aid visualization of RNA after electrophoresis
(Care should be taken on removal of the gel forming combs prior to loading of samples
as rupture of the well bottoms can occur. Agarose/formaldehyde gel is inherently weaker
than the equivalent percentage agarose gel).
5. Immediately prior to loading, RNA samples are heated to 65°C for 10 minutes to denature
any secondary structure, cooled on ice for 2 minutes and add 2 mL of sterile loading buffer.
6. Samples are loaded onto the gel and electrophoresed at 5 V/cm, with occasional buffer
recirculation, until the leading bromophenol blue dye front has migrated approximately
three quarters of the length of the gel.
7. Visualization of RNA is done by UV irradiation with short wave (254 nm) UV light.
Typical markers of RNA quality are 18S (~ 1900 bases) and 28S (~ 4800 bases) RNA
molecules.
Buffers
(i) 10X 3-morpholinopropanesulphonic acid (MOPS) buffer
MOPS – 0.2 M
Sodium acetate – 50 mM
EDTA – 5 mM
The buffer is adjusted to pH 7.0 with 1M NaOH and sterilised by autoclaving.
(ii) RNA denaturing buffer
Deionized formamide – 100% (10 mL)
Formaldehyde – 40% (3.5 mL)
10X MOPS buffer – 1.5 mL
Formamide is deionized by stirring 100 mL with approximately 20 g of Amberlite MB3
(or MB1) ion exchange resin for 15 minutes.
Electrophoresis of Nucleic Acids "'
REFERENCES
Brody, J.R., E.S. Calhoun, E. Gallmeier, T.D. Creavalle and S.E. Kern (2004), Ultra-fast high-
resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media,
Biotechniques, 37, pp. 598–602.
Milan, Bier (Ed.) (1959), Electrophoresis, Theory, Methods and Applications, 3rd ed., Academic
Press, p. 225.
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
# Laboratory Manual for Genetic Engineering
8
Slot Lysis Agarose Gel Electrophoresis
The slot lysis gel electrophoresis is an improved technique in which the presence of plasmid DNA
in bacterial colonies/clones can be directly analysed without isolating plasmid DNA. Through this
technique, the plasmid loss during plasmid purification could be avoided because the slots in the
gel are loaded with spheroplast and lysed in slots and the contents are electrophoresed subse-
quently. The DNA bands could be visualized after staining with ethidium bromide. Slot lysis gel
electrophoresis could be carried out both horizontally and vertically. Horizontal slot lysis gel
electrophoresis can be used for analyzing the plasmid profiles of gram negative bacteria such as
E. coli, Agrobacterium, Rhizobium, etc. Vertical slot lysis electrophoresis is most suitable for
analyzing the complex plasmid profiles of gram positive bacteria such as Bacillus thuringiensis,
B. megaterium, B. cereus, B. subtilis, B. sphaericus, etc. For both types of slot lysis gel elec-
trophoresis, TBE buffer is used and are run with different electrophoretic conditions. Vertical slot
lysis gel electrophoresis is the only technique that is used for the analysis of large sized mega
plasmids in Bacilli strains.
8.1 HORIZONTAL SLOT LYSIS ELECTROPHORESIS FOR E. COLI

Protocol
1. Bacterial cells from overnight grown colonies on agar plates are resuspended in
protoplasting buffer (15 mL) to a density of 104 or 105 cells/mL. Cells are mixed
thoroughly by vigorous vortexing. The mixture is incubated at 37°C for 10–15 minutes.
2. 0.8% agarose gels of needed slots are to be poured with 1X TBE buffer with 0.05%
SDS.
3. Preload the gel slots with 20 mL of lysis buffer. Allow to stand for 10–20 minutes.
4. Then load 10 mL of the protoplast suspension into each slot and carry out the
electrophoresis in 1X TBE buffer with 0.05% SDS.
5. Electrophoresis is carried out initially at 20 volts for 1 hour and for an additional 2 hours
at 120 volts.
6. After the completion of electrophoresis, stain the gel in 0.5 mg/mL ethidium bromide and
check for plasmid bands under UV trans-illumination and photograph.
50
Slot Lysis Agarose Gel Electrophoresis #
Buffers
(i) Protoplasting buffer (pH 8.0)
Tris-HCl – 30 mM
Na2 EDTA – 5 mM
NaCl – 50 mM
Sucrose – 20%
RNase A – 50 mg/mL
Lysozyme – 50 mg/mL
(ii) TBE-SDS buffer (pH 8.3)
Tris – 89 mM
Boric acid – 89 mM
Na2 EDTA – 2.5 mM
SDS – 0.05%
(iii) Lysis buffer
Tris – 89 mM
Na2 EDTA – 2.5 mM
SDS – 2%
Sucrose – 5%
Bromophenol blue – 0.04%
8.2 VERTICAL SLOT LYSIS ELECTROPHORESIS FOR

B. THURINGIENSIS (MODIFIED ECKHARDT’S LYSATE
ELECTROPHORESIS)
Protocol
1. Streak Bacillus sp. cells on plate of SCG or SCGY agar and incubate at 30°C for
10–16 hours depending on the growth capability of the strain.
2. Resuspend an inoculation loop full of cells (107–108 cells) from each streak in 50 mL of
lysozyme mixture (protoplasting buffer) by vortexing vigorously for 2 minutes.
3. Incubate the cells suspension in a 37°C water bath for 30–120 minutes depending on the
strain to generate sphaeroplasts.
4. Monitor the protoplast formation by phase contrast microscopy. Make sure that 99% of
sphaeroplasts are formed.
5. Pipette into each slot 20 mL of SDS lysis buffer to a vertical agarose gel prepared in
1X TBE buffer (dimensions: 125 mm, long 150 mm wide, 3 mm thick) and allow it to
stand for 20 minutes.
6. Pipette 10 mL of sphaeroplast suspension in protoplasting buffer under the SDS lysis
buffer without mixing the two layers.
# Laboratory Manual for Genetic Engineering
7. Start the electrophoresis immediately after loading the protoplasts.

8. Electrophoresis is done usually at a constant current of 3.0 mA for 1½ – 2 hours,
followed by a change to a constant voltage; first to 40 volts (8 – 10 mA) for 50 minutes,
then to 120 V (25 – 29 mA) for another 2½ to 3½ hours.
9. The gel slab is prevented from slipping between the glass plates of the apparatus by a
sponge and a 25 mL plug of 2% agarose.
10. After electrophoresis, stain the gel with 0.5 mg/mL ethidium bromide and check for the
plasmid profile of the organism under UV transilluminator and it can also be
photographed (Figures 8.1 and 8.2).
MDa MDa
Lanes: 1, B. thuringiensis
subsp. thuringiensis HD2
(marker strain); 2, HD977
wild type; 3, transductant
B. thuringiensis subsp.
yunnanensis (pBC16).
Figure 8.1 Slot lysis electrophoretic analysis of B. thuringiensis subsp. yunnanensis and
B. thuringiensis subsp. yunnanensis (pBC16). The molecular masses (in MDa)
of the small plasmids of strains HD2 and HD977 are shown on the left and right,
respectively.
Buffers
(i) Lysozyme mix or protoplasting buffer (10 mL)
Lysozyme – 2 mg/mL
20% sucrose – 2.0 g
100 mg/mL preboiled RNase A – 50 mL of RNase A stock (from 20 mg/mL)
30 mM Tris base – 0.03633 g
UV
W
5 mM Na2 EDTA – 0.01861 g pH 8.0
50 mM NaCl – 0.02922 g
Slot Lysis Agarose Gel Electrophoresis #!
Lanes: 1, B. thuringiensis subsp.

thuringiensis HD2 (marker strain);
2, B. thuringiensis subsp.
yunnanensis (pBC16), 3, HD73-26,
4, 5, and 6, B. thuringiensis
subsp. kurstaki trans-conjugants
73-26-13, 73-26-16, and 73-26-20,
respectively.
Figure 8.2 Slot lysis electrophoresis of B. thuringiensis strains showing megaplasmid. The
molecular masses (in MDa) of plasmids of strains HD2 and HD977 are shown on
the left and right, respectively.
(ii) Lysis buffer or SDS mixture (pH 8.3 – 8.5)

SDS – 2%
Sucrose – 5%
Bromophenolblue – 0.05%
Tris base – 89 mM
Na2 EDTA – 2.5 mM
(iii) Spizizen’s minimal medium (1 litre) (10X)
K2HPO4 – 152.0 g
KH2PO4 – 48.0 g
Na citrate – 10.0 g
(NH4)2SO4 – 2.0 g
Make up to 1 litre with dH2O. Add 1 mL of 1 M MgSO4 ◊ 7H2O to 1 litre of medium after
autoclaving.
(iv) 1 M MgSO47H2O (100 mL)
MgSO47H2O – 24.64 g
#" Laboratory Manual for Genetic Engineering
(v) SCG plates (1000 mL)

10X Spizizen’s salts without Mg 2+ – 100 mL
Bacto agar – 15 g
U| Autoclave and then add
Casamino acids – 1g V| Glucose and MgSO stock solutions
4
Make up to with H2O to – 979 mL W
25% glucose (stock solutions) – 20 mL
1 M MgSO47H2O (stock solutions) – 1 mL
Mix and pour onto plates
REFERENCES
Gonzalez, J.M., Jr., H.T. Dulmage and B.C. Carlton (1981), Correlation between
specific plasmids and delta endotoxin production in Bacillus thuringiensis, Plasmid 5,
pp. 351–365.
Sekar, V., D.V. Thompson, M.J. Maroney, R.G. Bookland and M.J. Adang (1987), Molecular
cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis
var. tenebrionis. Proc. Natl. Acad. Sci. 84, pp. 7036–7040.
Purification of DNA from Agarose and Polyacrylamide Gels ##
9
Purification of DNA from Agarose and
Polyacrylamide Gels
Gene-cloning experiments becomes easier when the DNA fragment of known size with the gene
of interest is purified from gel. When these DNA fragments are purified or eluted from agarose
or polyacrylamide gel they would be free from any other contaminating DNAs. The gel purified
DNA fragments are used for ligation during gene cloning, one could easily get the right clones
and the cumbersome job of screening a large number of clones could be avoided. Currently, there
are several companies that are marketing gel elution kits that are tailor-made to be used with normal
agarose gel slices. The user has to strictly follow the guidelines and methods of the respective
companies to get the optimum DNA recovery from agarose gels. This chapter explains conven-
tional methods of DNA elution from low melting point agarose gels and polyacrylamide gels.
9.1 ISOLATION OF DNA FROM AGAROSE GELS

Protocol
1. Run the restricted DNA samples along with a molecular weight-marker on a low melting
agarose gel. Stain the gel briefly with 0.5 mg/mL ethidium bromide stain and locate the
DNA band to be excised by a brief exposure to UV in a trans-illuminator. Excise the DNA
band using sterile razor and place the excised DNA-containing agarose gel slice in a
1.5 mL microcentrifuge tube. Freeze at –70°C for at least 15 minutes, or until frozen.
It is possible to pause at this stage in the elution procedure and leave the gel slice frozen
at –70°C.
2. Thaw the slice by incubating the tube at 65°C.
3. Add one-volume of TE-saturated phenol, vortex for 30 seconds and freeze the sample
at –70°C for 15 minutes.
4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at
room temperature to separate the phases. The aqueous phase is then removed to a clean
tube, extracted twice with equal volume ether, ethanol precipitated and the DNA pellet
is rinsed, dried and resuspended in less volume of TE buffer.
55
#$ Laboratory Manual for Genetic Engineering
9.2 ISOLATION OF DNA FRAGMENTS FROM POLYACRYLAMIDE

GELS
Protocol
1. Run and stain polyacrylamide gel as described in Section 7.2 of Chapter 7.
2. Use a long-wave UV lamp to locate the band of interest.
3. Cut out the band, using a sharp sterile razor.
4. Place the gel-slice on a glass plate and chop it into fine pieces with a razor blade.
5. Transfer the pieces to a small Eppendorf tube and add one volume of elution buffer.
6. Cap the tube and incubate at 37°C overnight, if possible on a rotating wheel.
7. Centrifuge the sample at 10,000 rpm for 10 minutes at 20°C. Recover the supernatant.
8. Be careful to avoid transferring fragments of acrylamide.
9. Add an additional 0.5 volume of elution buffer to the pellet, vortex briefly and recentrifuge.
Combine the two supernatants.
10. Remove any remaining fragments of acrylamide by passing the supernatant through a
sterile sieve by adding 0.3 M sodium acetate (pH 4.6).
11. Precipitate the DNA by adding 4 volumes of ethanol.
12. Redissolve the DNA in 200 mL of TE.
13. Add 25 mL of 3 M sodium acetate (pH 4.6) and reprecipitate the DNA with four volumes
of ethanol.
14. Wash the DNA pellet with 70% ethanol, air dry the pellet and redissolve in less volume
of TE buffer (pH 8.0).
Buffer
(i) Elution buffer
Ammonium acetate – 0.5 M
EDTA (pH 8.0) – 1 mM
REFERENCE
Grey, M. and M. Brendel (1992), Rapid and simple isolation of DNA from agarose gels, Current
Genetics, 22 (1), pp. 83–84.
Transformation of GRAM Negative and GRAM Positive Bacteria with Plasmid DNA #%
10
Transformation of GRAM Negative and
GRAM Positive Bacteria with Plasmid DNA
Although Miescher (1871) discovered nucleic acids in living cells, its central role in genetics was
not certain for another 70 years. Several important experiments were performed in the first half
of the 20th century that led the way to the identification of DNA as the genetic material. This
experiment provided the first strong evidence that DNA was the chemical basis of heredity. This
natural transformation involves DNA-binding proteins that limit the uptake of DNA to molecules
that are similar to those of the cell, typically DNA from the same species of bacterium. These
naturally transformed (competent) bacterial cells can also pick up and maintain small, circular
DNA molecules called plasmids that are self-replicating and remain independent of the host cell
chromosome. Plasmids are foreign DNA molecules, found in bacteria and yeasts, which can
contain genetic information from any source. Plasmids can cause transformation because
expression of the information in the plasmid DNA can change the phenotype of the bacterial cells
that contain them. Most bacteria are not capable of natural transformation, but can be induced
to pick up DNA from their environment, if treated to make the cell membrane permeable to DNA.
Escherichia coli (the bacterium naturally found in the human intestine) is not naturally
transformable (this is termed non-competent) but can be induced to take up foreign DNA from
the environment by treating with calcium ions and increasing temperature, or a high voltage
electrical field (electroporation). Using these inducing techniques, non-competent bacterial cells
can be induced to take up and incorporate DNA of their own species or plasmids. This chapter
deals with both the conventional transformation techniques such as the competent cell method
for E. coli and Bacillus sp. and the protoplast transformation method for Bacillus sp. and the
electroporation method of transformation for E. coli and Bacillus sp.
10.1 COMPETENT CELL TRANSFORMATION OF

GRAM NEGATIVE BACTERIA
Protocol
1. Inoculate 100 mL of Luria broth in a 500 mL flask with 1 mL of overnight culture of
E. coli grown from a single colony. Grow cells with vigorous shaking at 37°C to a
57
#& Laboratory Manual for Genetic Engineering
density of ~5 ¥ 107 cells/mL. This usually takes 2 to 4 hours. For each transformation
assay 3 mL of the cells will be needed.
2. Chill the culture on ice for 10 minutes. Centrifuge the culture at 4000X g for 5 minutes
at 4°C.
3. Discard the supernatant. Resuspend the cells in half of the original culture volume of an
ice-cold, sterile solution of 50 mM CaCl2 and 10 mM Tris-Cl (pH 8.0).
4. Place the cell suspension in an ice-bath for 15 minutes and then centrifuge the
suspension at 4,000X g for 5 minutes at 4°C.
5. Discard the supernatant. Resuspend the cells in 1/15th of the original volume in an ice-
cold sterile solution of 50 mM CaCl2 and 10 mM Tris-Cl (pH 8.0). Dispense 0.2 mL
aliquots into pre-chilled tubes. Store the cells at 4°C or in ice for 12 to 24 hours.
6. Add DNA to be transformed in ligation buffer or TE buffer. Mix and store on ice for
30 minutes. Up to 40 ng of DNA (dissolved in up to 100 mL of ligation buffer or TE
buffer) can be used for each transformation reaction (Addition of more DNA or a greater
volume of buffer leads to a reduction in transformation efficiency).
7. Transfer to a water bath, preheated to 42°C for 2 minutes.
8. Add 1 mL of fresh Luria broth to each tube and incubate at 37°C for 30 minutes
(tetracycline selection) or 1 hour (ampicillin or kanamycin selection) without shaking.
This period allows the bacteria to recover and to begin to express antibiotic resistance.
9. Spread an appropriate quantity of cells (minimum of 100 mL) onto selective medium using
either the spreading or top agar procedure. Usually the top agar procedure yields slightly
more transformant (The entire transformation mixture may be spread on a single plate
or plated in top agar, if the selection is for tet resistance. If ampicillin resistance is
required, only a portion of the culture should be spread).
10. Leave the plates at room temperature in the laminar air flow system until the top agar
has hardened or until the liquid has been absorbed.
11. Incubate the plates at 37°C. Colonies should appear within 12 to 16 hours and score the
transformants.
Buffer
(i) Transformation solution (100 mL) (pH 8.0)
CaCl2 – 50 mM
Tris – 10 mM
10.2 E. COLI TRANSFORMATION BY CALCIUM

CHLORIDE METHOD
Protocol
1. Incubate 50 mL Luria broth with 0.5 mL of overnight culture of E. coli strain grown
from a single colony.
2. Grow the culture to an OD of 0.4 to 0.6 (at a wavelength of 600 nm).
3. Chill the culture in ice for 10 to 30 minutes and centrifuge at 6,000 rpm for 5 minutes
at 4°C and take the cell pellet by decanting the culture supernatant aseptically.
Transformation of GRAM Negative and GRAM Positive Bacteria with Plasmid DNA #'
4. Resuspend the cells in equal or 1/2 the volume of 0.1 M of CaCl2 (ice-cold). Keep in
ice for 20 to 30 minutes.
5. Centrifuge the cells again at 6,000 rpm for 5 minutes at 4°C and aseptically remove the
supernatant completely.
6. Resuspend the pellet in 1/10th volume 0.1 M CaCl2 (ice-cold) and keep it in ice for
1/2 hour to 1 hour (the efficiency of transformation increases till 20 hours of incubation
in ice cold CaCl2 and then decreases).
7. Take 100 mL of cell suspension and add diluted ligated mix and mix well and leave the
tube in ice for 30 minutes.
8. Heat shock at 42°C for 2 minutes exactly.
9. Incubate the tube again in ice for 3 minutes.
10. Add 0.9 mL of fresh Luria broth. Keep it at 37°C with gentle shaking for 1 hour.
11. Aliquots of transformed cells plate on selective medium.
12. Incubate the controls as shown in Table 10.1.
13. Incubate the plates at 37°C in incubator for 17 hours and score the transformants.
TABLE 10.1 The following controls will help to check the correct transformants
Controls Medium Result
Cut vector + ligase Selective Colonies should come
Cut vector – ligase Selective No colonies should come
Uncut vector (20 ng) Selective Colonies should come
Cells – DNA Selective No colonies should come
Viability (two dilutions) Non-selective Colonies should come
Transformed cell samples Selective Colonies should come
10.3 E. COLI TRANSFORMATION BY TSB BUFFER METHOD

Protocol
1. Inoculate 50 mL Luria broth with 0.25 mL of overnight culture of E. coli strain grown
from a single colony.
2. Grow the culture to an OD of 0.3 to 0.6 at a wavelength of 600 nm.
3. Pellet the cells at 6,000 rpm for 5 minutes at 4°C.
4. Resuspend the pellet in 0.5 mL of TSB buffer at ice-cold temperature. The cells can be
subsequently frozen (in a dry ice/ethanol bath) and can be stored at –70°C for future use.
5. Incubate the cells on ice for 10 minutes.
6. Take 0.1 mL of cells suspension and add 100 mg of DNA in an ice-cold Eppendorf tube.
7. Grow the cells at 37°C after adding 0.9 mL of TSB buffer with 20 mM glucose for
60 minutes at 225 rpm.
8. Plate aliquots of the cells on antibiotic supplemented agar medium.
9. Incubate the cells at 37°C incubator for 16 hours and score the transformants.
Buffer
(i) TSB buffer (transformation and storage buffer) 50 mL
Yeast extract – 0.25 gm
Tryptone – 0.50 gm
NaCl – 0.50 gm
PEG (3000) – 5.0 gm
Adjust the pH to 6.1, autoclave and add
DMSO – 0.5 mL
1 M MgSO4 – 50 mL
1 M MgCl2 – 50 mL
10.4 E. COLI TRANSFORMATION BY ELECTROPORATION

Protocol
1. Grow E. coli in Luria broth at 37°C with vigorous shaking.
2. Harvest the cells in mid log phase (ABS600 nm between 0.5 to 1.0).
3. To reduce ionic strength of the cell media and concentrate the cells, chill the cells on ice
briefly, then pellet at 4°C in a centrifuge at 400X g for 15 minutes. Resuspend in original
volume in sterile water (ice cold), pellet as above, resuspend the pellet in 0.5 volume of
sterile ice cold H2O, resuspend the pellet in 0.02 volume of sterile ice cold 10% glycerol
and resuspend in 0.02 to 0.003 volume of sterile ice cold 10% glycerol (resuspend in
as small volume as possible for maximum transformation efficiencies).
4. Pellet 60 mL aliquots into 0.5 mL centrifuge tubes and quickly freeze in liquid nitrogen
and store at –70°C for future use.
5. Sterilize the electrode with ethanol then chill on ice (the flat pack chamber P/N 486 is
placed on foil on ice, the cuvette electrode P/N 474 is placed in the cuvette on ice).
6. Switch on the BTX pulse power generator (or BioRad electroporator). Set the voltage
amplitude to 600–950 volts and set the pulse length to 5 m second.
7. Keep the cells on ice; add 2 mL of DNA (2 pg/mL to mg/mL range in low ionic strength
buffer) pipette twice to mix, then pipette 40 mL between electrodes.
8. Chill on ice for 45 seconds (if using the flatpack chambers, next slide it down into
flatpack holder, with the chamber, oriented horizontally, so that at least 1/8 to 1/4 of an
inch protrudes above the holder). Slide the flatpack holder on to the flatpack safety and
then pulse the cells by pressing the start or pulse button.
9. After electric pulse, quickly dilute and transfer cells by flushing the electrode with 1 mL
of SOC medium into a 17 ¥ 100 mm round bottom polypropylene tube.
10. Incubate at 37°C with constant agitation for 1 hour for expression of genes from the
acquired plasmid.
11. Then dilute the culture and plate on selective medium.
12. The best repeatable result is 1 ¥ 1010 transformants/mg DNA using 4 pg DNA and MC
1061 at 900 volts (14.4 kV/cm) and 5 m second pulse length.
Transformation of GRAM Negative and GRAM Positive Bacteria with Plasmid DNA $
Buffers
(i) Luria broth 1000 mL (pH 7.3)
Tryptone – 10 g
Yeast extract – 5 g
NaCl – 5g
(ii) 10% glycerol (100 mL)
Glycerol – 10 mL
90 mL water and filter sterilize
(iii) SOC broth (pH 7.3) (50 mL)
2% Bacto tryptone – 1.00 g
0.5% Bacto yeast extract – 0.25 g
10 mM MgCl2 – 0.10 g
10 mM MgSO4 – 0.12 g
20 mM glucose – 0.09 g
10 mM KCl – 0.04 g
0.06% NaCl – 0.03 g
10.5 SIMPLE METHOD OF PLASMID TRANSFORMATION OF

E. COLI BY RAPID FREEZING
Protocol
1. Exponentially growing bacterial cells should be used for transformation.
2. E. coli cells at a density of 0.2 to 0.4 OD at 600 nm are to be suspended in Luria broth
10 ng of circular plasmid DNA is to be mixed with 100 mL of E. coli cell suspension
in a 1.5 mL microfuge tube.
3. The mixture should be plunged directly into liquid nitrogen in a small container for
1 minute.
4. After freezing, E. coli cells are to be thawed at room temperature and then spread onto
selective agar plates and incubated at 37°C overnight.
5. Test for the presence of plasmids in the transformants.
10.6 PROTOPLAST TRANSFORMATION OF

BACILLUS SP. WITH PLASMID DNA
Protocol
1. Grow culture of Bacillus sp. at 30°C overnight on nutrient agar.
2. Inoculate single colony in 20 mL RHAF broth from overnight nutrient agar plate. Grow
cells at 37°C for 2 to 2½ hours.
3. Harvest the cells at room temperature using a table top centrifuge.
4. Resuspend pellet in 250 mL of HAF protoplasting buffer with 1 mg/mL lysozyme for
10 to 15 minutes. Check for protoplasts under phase contrast microscope. Ensure 99%
protoplasts.
5. Centrifuge at 2000 rpm for 10 minutes. Wash the pellet with RHAF medium.
6. Resuspend pellet in 1 mL of RHAF (use less volume to concentrate the protoplasts).
7. Add 1 mg DNA to the protoplasts in a polypropylene centrifuge tube and then add equal
volume of 30% PEG (6000) in HAF.
8. Incubate the tube at 37°C for 5 minutes.
9. Add 5 mL RHAF to dilute PEG.
10. Centrifuge at 2000 rpm and resuspend in 1 mL RHAF and incubate at 30°C for 2 hours
at slow shaking.
11. Plate aliquots of cells on appropriate antibiotic plates. Incubate at 30°C for 1 to 2 days.
Buffers
(i) HAF protoplasting buffer for 1000 mL (Isotonic minimal medium (1X)
NH4Cl – 1.0 g
Tris – 12.0 g
KCl – 0.035 g
NaCl – 0.058 g
Na2SO410H2O – 0.3 g
KH2PO4 – 0.14 g
MgCl2.5H2O – 4.26 g
Glucose – 2.0 g
Sucrose – 68.46 g
Note: Prepare the stock solution except MgCl2 5H2O, glucose and sucrose in 10X and
adjust the pH 7.5.
Prepare the stock solution of MgCl2 5H2O, glucose and sucrose individually.
(ii) MgCl25H2O
Add 1.15 mL of 2 M MgCl25H2O to 100 mL of medium RHAF.
(iii) Glucose
Add 0.4 mL of 50% glucose to 100 mL of RHAF medium.
(iv) Sucrose
Add 3.42 mL of 40% sucrose to 100 mL of RHAF growth medium.
(v) RHAF growth medium
Add 0.05% yeast extract.
0.05% Tryptone to HAF salt solution for broths.
(vi) RHAF agar medium
Add agar to a final concentration of 1% to RHAF growth medium (broth).
10.7 PROTOPLAST TRANSFORMATION OF BACILLUS

SPHAERICUS WITH PLASMID DNA
Protocol
1. Grow Bacillus sphaericus cells in Penassay broth at 37°C with shaking at 250 rpm until
mid-exponential phase of growth is achieved.
Transformation of GRAM Negative and GRAM Positive Bacteria with Plasmid DNA $!
2. Harvest the cells in room temperature and resuspend the cell pellet in 1/6th volume of
Sucrose Maleate Magnesium Chloride Penassay (SMMP) with 1% BSA.
3. Add lysozyme to a final concentration of 10 mg/mL (2 mg/mL for B. subtilis) and the
suspension is to be incubated at 37°C for 1 hour with gentle shaking.
4. Monitor protoplasting by phase contrast microscopy. Ensure 90% protoplasts.
5. Centrifuge the protoplasts at 2600 rpm for 15 minutes at room temperature. Wash the
pellet once with SMMP and resuspend the protoplasts in 1/15th volume of starting
culture in SMMP broth.
6. Mix 1 to 5 mg of plasmid DNA in 50 mL of TE buffer with equal volume of 2X SMM
solutions in a bacteriologically sterile culture tube. To which add 0.5 mL of protoplast
suspension in SMMP and immediately add 0.5 mL of 40% PEG solution in SMM and
gently mix the content of the tube.
7. Incubate for 5 minutes at 37% incubator shaker.
8. Add 5 mL of SMMP medium to the mixture to dilute the PEG and the protoplasts are
recovered by centrifugation at 2,600 rpm for 15 minutes at room temperature.
9. The treated protoplasts are now to be resuspended in 1 mL of SMMP with 1% BSA and
incubate at 30°C with gentle shaking for 2 hours for phenotypic expression of genetic
determinants carried by the transforming plasmid.
10. Plate appropriate dilution of the protoplasts suspension (after keeping for expression) on
antibiotics supplemented DM3 regeneration medium containing 0.23 M sodium
succinate. The medium can be altered with the substitution of glycerol for glucose.
11. Incubate the plates at 30°C for 2 to 5 days and examine for the transformants.
Buffers
(i) Penassay broth 1X for 1000 mL
Bacto-beef extract – 1.5 g
Yeast extract – 1.5 g
Peptone – 5.0 g
Dextrose – 1.0 g
Sodium chloride – 3.5 g
Dipotassium phosphate – 3.68 g
Monopotassium phosphate – 1.32 g
(ii) 2X SMM buffer (pH 6.5) 500 mL
Sucrose 1.0 M – 171.0 g
Na2 Maleate 0.04 M – 3.2 g
MgCl26H2O – 4.066 g
(iii) SMMP broth
Mix 50 mL of sterile 2X SMM buffer with 50 mL of sterile 4X Penassay broth in a sterile
bottle.
(iv) 40% PEG in SMM buffer
PEG6000 – 40 g
2X SMM – 50 mL
Dissolve and make up to 100 mL and sterilize.
$" Laboratory Manual for Genetic Engineering
(v) DM3 regeneration plate (1000 mL)

Bactoagar – 8.0 g in 200 mL (0.8%)
Na2 Succinate – 135.0 g in 500 mL (0.5M)
Casamine acid – 5.0 g in 100 mL (0.5%)
Yeast extract – 5.0 g in 50 mL (0.5%)
K2HPO4
KH2PO4
– 3.5 g
– 1.5 g } (0.5%) in 100 mL
It is necessary using large quantities of NaOH for adjusting pH to 7.3.
Autoclave separately and mix, keep at 55°C for 30 minutes and then add the following
sterile solutions to the mixed cooled medium at (55°C).
Glucose – 25 mL of 20%
MgCl2◊6H2O – 20 mL of 1 N
BSA (Filter sterilized) – 5 mL of 1%
Mix and pour on to the plates.
(vi) Modified DM3 regeneration plate (300 mL)
Bactoagar – 2.40 g in 60 mL
Sucrose (0.5 M) – 51.34 g in 150 mL
Casaminoacid – 1.5 g in 15 mL
}
Tryptone – 3.0 g in 30 mL
NaCl – 1.5 g
UV
W
K2HPO4 – 0.05 g in 22.5 mL
KH2PO4 – 0.45 g
Autoclave separately and then mix and cool to 55oC for 30 minutes and then add the
following:
Glucose – 15 mL of 10% stock
MgCl2 – 6 mL of 1 M stock
BSA – 1.5 mL of 1% stock
Mix and pour onto plates.
10.8 COMPETENT CELL TRANSFORMATION OF

BACILLUS SUBTILIS WITH PLASMID DNA
Protocol
1. Inoculate a 5 mL of SP-I medium with a single B. subtilis colony from a nutrient agar
plate and culture O/N. Incubate overnight at 37°C with rapid shaking.
2. Inoculate 50 mL of SP-I medium with 0.5 mL of the overnight culture; incubate at 37°C
with rapid shaking monitor growth by absorbance until stationary phase.
3. Add 5 mL of this early stationary phase culture to 45 mL of prewarmed SP-II medium.
Shake slowly at 37°C for 90 minutes.
4. Add 0.5 mL of EGTA and continue shaking at 37°C for 5–10 minutes.
Transformation of GRAM Negative and GRAM Positive Bacteria with Plasmid DNA $#
5. Add 0.1–0.5 mL of cell suspension to 1–3 mg of DNA in a small sterile polypropylene

tube and shake at 37°C.
6. After 90 minutes of incubation, plate 0.05–0.1 mL onto dry agar plates containing
appropriate antibiotics. Incubate at 37°C for 2 days and score the transformants.
7. To freeze cells for later use do not add EGTA after step 4, cool on ice and harvest by
centrifugation (6,500 rpm/4°C, 10 minutes).
8. Gently freeze the cell suspension rapidly in dry ice-ethanol. Store at –70°C (for storage
of cells for future also).
9. To prepare the transformation, thaw the cells rapidly at 42°C. Add 4 mL SP-II medium
per mL of cells.
10. Add 1/100 volume of EGTA, wait for 5 minutes and transform as in steps 5 and 6.
Buffers
(i) SP-I salts 250 mL
(NH4)2SO4 – 0.5 g
U|
K2HPO4 – 3.5 g
V|Filter sterilize
W
KH2PO4 – 1.5 g
Na citrate 2H2O – 0.25 g
MgSO4◊7H2O – 0.05 g
(ii) CaCl2
CaCl2 – 0.28 g
(iii) MgCl2
MgCl2 – 2.54 g
Hot distilled water up to 50 mL
(iv) Glucose
Glucose – 25 g
(v) CAYE
Casaminoacids – 1.0 g
(vi) EGTA
Ethylene Glycol-bis (b-aminoethyl ether)-N-N’-tetra acetic acid-1.9 g
(vii) SP-I medium
SPI salt – 100 mL
Glucose – 1 mL
CAYE – 1 mL
$$ Laboratory Manual for Genetic Engineering
(viii) SP-II medium

SPI salts (stock) – 100 mL
Glucose (stock) – 1 mL
CAYE (stock) – 1 mL
CaCl2 (stock) – 1 mL
MgCl2 (stock) – 1 mL
10.9 TRANSFORMATION OF BACILLUS THURINGIENSIS

BY ELECTROPORATION
Protocol
1. Grow B. thuringiensis cells to an OD of 0.5 at the absorbance at the wavelength of
600 nm.
2. Pellet the cells for electroporation without pulse checker, wash the cells in the sucrose
electroporation buffer and resuspend in 1/20th of the original volume (7 ¥ 108 CFU/mL)
of sucrose electroporation buffer.
3. For electroporation with pulse checker: Wash twice with water and once with HEPES-
glycerol electroporation buffer and resuspend in 1/20th volume of this buffer (to the
maximum of 109 to 1010 CFU/mL).
4. 0.8 mL of cells suspended in sucrose phosphate buffer (SPB) or HEPES-Glycerol (HG)
buffer and chilled on ice in a 0.4 cm gene pulser cuvette and 500 ng of DNA in 5 mL
TE is to be added to the cells and mixed.
5. After a maximum of 10 minutes incubation in ice, the cells are to be subjected to a
single pulse (capacitance 25 mF, set voltage 1.8 to 2 kV, initial field strength 4,500 to
5,000 V/cm in 5 m seconds).
6. After the pulse the cells are to be returned to ice for up to 10 minutes, then 5.6 mL of
Luria broth to be added and incubated at 30°C for 1 hour to allow expression of
antibiotics resistance, prior to plating on LB agar plates containing suitable antibiotics.
7. Incubate the plates at 37°C incubator for 16 hours and score the transformants.
Buffers
(i) Sucrose electroporation buffer (SPB buffer) (pH 7.2)
Sucrose – 272 mM
Sodium phosphate buffer – 7 mM
MgCl2 – 1 mM
(ii) HEPES-Glycerol electroporation buffer (HG buffer)
HEPES (N-2-Hydroxyethyl-piperazine- N’-2-ethane-sulphonic acid) pH 7.0 – 1 mM
Glycerol – 10%
(iii) Preparation of phosphate buffer
Stock solutions
A: 0.2 M solution of monobasic sodium phosphate (27.8 g in 1000 mL)
Transformation of GRAM Negative and GRAM Positive Bacteria with Plasmid DNA $%
B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4:7H2O or 71.7 g of

Na2HPO4:12H2O in 1000 mL)
X mL of A + Y mL of B, mixed and diluted to a total of 200 mL to get the required fixed
pH (Table 10.2).
TABLE 10.2 Preparation of phosphate buffers with specific pH

X Y pH
93.5 06.5 5.7
92.0 08.0 5.8
90.0 10.0 5.9
87.7 12.3 6.0
85.0 15.0 6.1
81.5 18.5 6.2
77.5 22.5 6.3
73.5 26.5 6.4
68.5 31.5 6.5
62.5 37.5 6.6
56.5 43.5 6.7
51.0 49.0 6.8
45.0 55.0 6.9
39.0 61.0 7.0
33.0 67.0 7.1
28.0 72.0 7.2
23.0 77.0 7.3
19.0 81.0 7.4
16.0 84.0 7.5
13.0 87.0 7.6
10.5 90.5 7.7
08.5 91.5 7.8
07.0 93.0 7.9
05.3 94.7 8.0
REFERENCES
Belliveau, B.H. and J.T. Trevors (1989), Transformation of Bacillus cereus vegetative cells by
electroporation, Appl. Environ. Microbiol, 55(6), pp. 1649–1652.
Chung, C.T., S.L. Niemela and R.H. Miller (1989), One-step preparation of competent
Escherichia coli: transformation and storage of bacterial cells in the same solution, Proc Natl
Acad Sci., 86(7), pp. 2172–2175.
Dower, W.J., J.F. Miller and C.W. Ragsdale (1988), High efficiency transformation of
Escherichia coli by high voltage electroporation, Nucleic Acids Res., 16, pp. 6127–6145.
Dubnau, D. (1999), DNA uptake in bacteria, Annu. Rev. Microbiol, 53, pp. 217–244.
$& Laboratory Manual for Genetic Engineering
McDonald, K.O. and W.F. Burke, Jr. (1984), Plasmid transformation of Bacillus sphaericus
1593, J Gen Microbiol, 130 (1), pp. 203–8.
Michel, B., B. Niaudet and S.D. Ehrlich (1982), Intramolecular recombination during plasmid
transformation of Bacillus subtilis competent cells, EMBO J, 1(12), pp. 1565–1571.
Takahashi, R., S.R. Valeika and K.W. Glass (1992), A simple method of plasmid transformation
of E. coli by rapid freezing, Biotechniques, 13(5), pp. 711–2, 715.
Estimation of Proteins $'
11
Estimation of Proteins
Proteins, one of the most important macromolecules found in all living cells, are nothing but
polymers of different amino acids (the word protein was derived from the Greek word proteios
which means, of primary importance, coined by J.J. Berzelius in 1838). There are about twenty
different amino acids, which make these proteins. These amino acids are organic, amphoteric
molecules having an amino group at one end and a carboxylic group at the other end. In proteins,
the amino acids are connected with peptide bonds.
Various methods, such as Biuret, UV, Lowry, Bradford, Dye binding and fluorescamine, are
available to determine the concentration of proteins in the biological samples. Each of these
methods vary in their sensitivity and hence, their applicability. Among these above-mentioned
methods, Bradford’s and Lowry’s Methods are frequently used for protein quantitation in most
of the laboratories.
11.1 ESTIMATION OF PROTEIN BY BRADFORD’S METHOD

This has also become a popular method for estimation of proteins because the assay is simple, quick
and inexpensive. This method is based on the principle that proteins bind to Coomassie brilliant
blue G-250 in acid solution and form a complex whose extinction coefficient (l max = 595 nm)
is much greater than the free dye (lmax = 465 nm) itself.
The Coomassie brilliant blue G-250 also known as Serva blue-G appears as a pale-orange
red in protonated form, i.e. in acid solution. The dye binds strongly to positively-charged groups
of proteins (especially to Arg residues and to a lesser extent to Lys, His and aromatic amino acids)
and also to hydrophobic regions in proteins. As a result, a blue colour is formed with a l max at
595 nm. (On binding to proteins the l max is shifted from 465 to 595 nm).
Protocol
Protein assay:
1. Add 5 mL of the Bradford’s reagent to 100 mL of test solution containing 10–100 mg
protein. Mix well and leave for 2 minutes and read the absorbance at 595 nm.
69
% Laboratory Manual for Genetic Engineering
2. The reading of absorbance should be plotted on a standard graph with BSA (1 mg/mL)
stock and from standard graph the unknown protein can be estimated.
Preparation of a standard graph

Prepare a standard graph with BSA from 10 mg to 100 mg (from 1 mg/mL stock) plot the protein
weight in X-axis and the OD in Y-axis. The line must be straight and should connect at least
3 points.
Buffers
(i) Bradford reagent 1000 mL
Dissolve 100 mg of Coomassie brilliant blue G 250 in 50 mL, 95% ethanol for 1000 mL
reagent.
Add 100 mL of 85% (w/v) phosphoric acid.
Make up to 1 L with water.
Specific gravity of phosphoric acid 1.75 g: 1 mL of phosphoric acid = 1.75 g,
85 g = 48.6 mL.
85% = 48.6 mL. Make up to 100 mL (4.86 mL to 10 mL, 9.72 mL to 20 mL).
(ii) Protein dissolving buffer (pH 9.5 to 10.5)
Sodium bicarbonate – 50 mM
DTT – 10 mM
NaOH (pH 10.5) – 10 mM
DTT – 25 mM
11.2 ESTIMATION OF PROTEIN BY LOWRY’S METHOD

This is most widely used method in all laboratories. In this method, the proteins react with the
Folin’s phenol reagent and produce a blue colour. The colour development relies on the following:
1. The formation of a copper-protein complex.
2. The reduction of phosphomolybdate and phosphortungstate anions present in folin-
ciocalteu phenol reagent by the tyrosine and tryptophan residues of the proteins, to
heteropolymolybdenum blue and tungsten blue respectively, this gives a blue-coloured
complex with a l max of 750 nm.
3. Cu2+ also acts as a catalyst in the reduction reaction.
The intensity of the colour mainly depends on the aromatic amino acid content of a protein.
Approximately 75% reduction occurs due to the copper protein complex with tyrosine and to
lesser extent (25%) with tryptophan residue.
Protocol
1. 0.1 mL or 0.01 mL sample is made up to 1 mL with distilled water.
2. Add 4 mL of alkaline copper reagent.
3. Keep it in dark for 15 minutes.
4. Add 0.5 mL of diluted folin’s reagent.
Estimation of Proteins %
5. Keep it in dark for another 20 minutes.

6. Read the absorbance at 750 nm.
The reading of absorbance is to be plotted on a standard graph with BSA (1 mg/mL) stock
and estimate the protein.
Preparation of standard graph

Prepare a standard graph with BSA from 10 mg to 100 mg (from 1 mg/mL stock). Plot the protein
weight in X-axis and the OD in Y-axis. The line must be straight connecting at least 3 points.
Buffers
Solution A: 2% Sodium carbonate in 0.1 N (1 g of Na2CO3 in 50 mL of 0.1 N).
Solution B: 1% CuSO4 (0.1 g of CuSO4 in 10 mL of d.H2O).
Solution C: 2% Sodium potassium tartrate (0.2 g of NaK tartrate in 10 mL of d.H2O).
(i) Alkaline copper reagent: Prior to use 50 mL of solution A is mixed with 0.5 mL each
of solution B and C.
(ii) Folin reagent: 3 mL of folin reagent is diluted with 3 mL of d.H2O (1:1 ratio).
REFERENCES
Bradford, M.M. (1976), A Rapid and Sensitive Method for the Quantitation of Microgram
Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem, 72,
pp. 248–254.
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951), J.Biol.Chem 193,
p. 265.
% Laboratory Manual for Genetic Engineering
12
Sodium Dodecyl Sulphate Polyacrylamide
Gel Electrophoresis for Proteins
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), is a technique widely

used in biochemistry, forensics, genetics and molecular biology to separate proteins according
to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight
as well as higher order protein folding, post-translational modifications and other factors). By
heating the protein samples under denaturing and reducing conditions, proteins become unfolded.
SDS-PAGE uses an anionic detergent (SDS) to denature proteins. The protein molecules become
linearized. One SDS molecule binds to 2 amino acids. Due to this, the charge to mass ratio of
all the denatured proteins in the mixture becomes constant. These protein molecules move in the
gel (towards the anode) on the basis of their molecular weights and are separated.
The charge to mass ratio varies for each protein (in its native or partially denatured form).
Mercaptoethanol assists the protein denaturation by reducing all disulphide bonds. The gel matrix
is formed of polyacrylamide. The polyacrylamide chains are cross-linked by N, N-methylene
bisacrylamide comonomers. Polymerisation is initiated by ammonium persulphate (radical
source) and catalysed by TEMED (a free radical donor and acceptor). The resolution and focus
of the protein bands is increased by using discontinuous gels (Laemmli gels)—the stacking gel
(pH 6.8, %T = 3 to 5 %) and the resolving gel (pH 8.8, %T = 5 to 20 %). %T represents
acrylamide percentage. These gels are usually run at constant current. At pH = 6.8, most of the
glycine in the population exist as zwitterions with no negative charge (pKa 1 = 2.45; pKa 2 =
9.6; pI = 6.025). Only 0.0015% of the glycine is anionic at this pH (refer glycine titration curve
and Henderson-Hasselbach equation). As such, bulk of the current is carried by the denatured,
negatively charged, SDS-coated protein molecules. At this stage, the glycine ions lag behind the
proteins. The order is as follows—chloride ions, denatured proteins, glycine ions.
Upon entering the resolving gel (pH = 8.8), the glycine deprotonate to the anionic form. The
proportion of these ions increases from 0.0015% to 15.8%. The carrying of the current is now
shared by the ions such that protein molecules have a greater freedom to separate on the basis
of molecular weights. Due to their small size, the glycine anions also tend to overtake the protein
band, thus providing a sandwiching effect and greater resolution in the gel.
72
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Proteins %!
12.1 SODIUM DODECYL SULPHATE–POLYACRYLAMIDE

GEL ELECTROPHORESIS OF PROTEINS
Protocol
1. Take out the gel solutions from the fridge and leave them at room temperature.
2. Clean the glass plates after soaking overnight in a detergent. Wash thoroughly with water
and dry it in hot air oven.
3. Place the notched plate on a paper. Apply the vacuum grease and fix three 1.0 mm
plexiglass spacer strips on both sides and the bottom. Place the regular plate on the
spacers and clip them on both sides and bottom, with 6 clips (2 on each side).
4. Mix the separating gel solutions gently omitting SDS and APS (Tables 12.1 and 12.2).
Degas the solution.
TABLE 12.1 Long gel receipe

Separating gel: 40 mL Stacking gel: 10 mL
Acrylamide: Bisacrylamide = 13.32 mL Acrylamide: Bisacrylamide = 01.34 mL
Separating gel buffer pH 8.8 = 10.00 mL Stacking gel buffer pH 6.8 = 02.5 mL
10% SDS = 00.40 mL 10% SDS = 00.1 mL
dd.H2O = 16.00 mL dd.H2O = 06.0 mL
Ammonium persulphate = 00.20 mL Ammonium persulphate = 00.05 mL
TEMED = 20 mL TEMED = 04 mL
Final volume = 40 mL Final volume = 10 mL
TABLE 12.2 Mini gel receipe

Separating gel: 10 mL Stacking gel: 5 mL
Acrylamide: Bisacrylamide = 03.23 mL Acrylamide: Bisacrylamide = 00.67 mL
Separating gel buffer pH 8.8 = 02.50 mL Stacking gel buffer pH 6.8 = 01.25 mL
10% SDS = 00.10 mL 10% SDS = 50.00 mL
dd.H2O = 04.00 mL dd.H2O = 03.0 mL
Ammonium persulphate = 50.00 mL Ammonium persulphate = 25.00 mL
TEMED = 05.00 mL TEMED = 02.00 mL
Final volume = 10.00 mL Final volume = 05.00 mL
5. Now add SDS and take ~ 1.0 mL of the separating gel solution in a small test tube and
add 20 mL APS. Leave it for 5–10 minutes. Check whether it has polymerized. If it has
polymerized proceed to the next step.
6. Take 1.0 mL of the separating gel solution and add 20 mL APS and quickly through the
inner edges of the plate. This will polymerize immediately and seal the bottom.
7. Now add the required volume of APS for the rest of the solution and pour the solution
into the glass plate using a pipette (do not mouth pipette) up to a level of about 4.0 cm
from the top (this distance can be determined beforehand by using the comb and the gap
required for the stacking gel).
%" Laboratory Manual for Genetic Engineering
8. Using a Pipetman, layer carefully on the gel with gel layering solution through the inner
edges one side, without disturbing the surface of the gel. Layering helps to form a
smooth, even gel surface and also excludes oxygen on the surface (oxygen inhibits gel
polymerization).
9. Allow the gel to stand for 30 minutes to 60 minutes to polymerize. A clear butanol—gel
interphase will be visible when the solution gets polymerized.
10. Once the gel is polymerized, pour off the top layer by tilting the gel, wash the gel surface
gently with distilled water, wipe dry the surface and inside of the glass plate with
Whatman No. 1 filter paper strips or tissue paper without touching the gel surface.
11. Mix the gel solutions gently for stacking gel as given above.
12. Rinse the top of the gel with ~1.0 mL of stacking gel solution and pour off.
13. Fill the top of the gel with stacking solution.
14. Insert the comb gently between the gel plates. Take care not to trap any air bubbles
below the teeth of the comb.
15. Allow the gel to stand for at least 30 minutes to polymerize (Opaqueness indicates the
polymerization of the gel.
16. Remove bottom clips and the bottom spacer. Remove the comb gently by sliding
vertically upwards. Rinse the wells gently through the sides of the plate with distilled
water and invert to drain the wells.
17. Apply vacuum grease and fix the glass plate with the gel tank tightly with clips on both
the sides. Make sure the buffer from the upper tank does not leak (Leaks can be sealed
with 1% molten agar).
18. Fill the upper and lower chambers with running buffer. Connect leads to the DC power
supply. The cathode (black terminal) of the upper chamber is connected to the black
terminal of power supply and the red terminal of the bottom chamber is connected to
the red.
19. Prepare sample solutions by mixing (1:1) in 2X sample buffer by placing the tube for
3 minutes in a boiling water bath. Cool to room temperature, spin for 1 minute and load
using micropipettes. 10 to 40 mL of sample containing ~25 mg of total proteins can be
loaded. Preferably do not load on the first and last well.
20. Load protein molecular weight standards in one of the wells.
21. Using a syringe with bent needle, remove air bubbles (if any) under the gel between the
glass plates in the bottom tank.
22. Electrophoresed the gel at 80 volts until the tracking dye has reached ~ 0.5 cm from the
bottom of the gel.
23. Turn-off power supply, disconnect the leads and remove the glass plates from the tank.
24. Place the plate on the bench, remove the side spacers and open the plates gently with
a spatula. Now the gel will be on the bottom plate. Gently flush water between gel and
the glass plate using a syringe and needle. Now the gel will be loosened on the plate.
25. Gently slide the gel into a tray containing the stain solution.
26. Stain the gel for 2–3 hours or overnight at room temperature.
27. Pipette out the staining solution and replace with the destaining solution (Table 12.3).
Change the destaining solution 2 to 3 times.
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Proteins %#
TABLE 12.3 Destaining solutions
50% methanol 5% methanol
10% acetic acid 7% acetic acid
Methanol 500 mL 50 mL
Acetic acid 100 mL 70 mL
dd.H2O 1000 mL 1000 mL
28. After complete destaining, the gel is stored in 10% acetic acid and photographed
(Figure 12.1). The gel can also be dried on a gel dryer for autoradiography.
kDa kDa
Lanes: 1, Prestained protein
molecular mass standards (top to
bottom: myosin, phosphorylase b,
bovine serum albumin, ovalbumin,
carbonic anhydrase, and b-
lactoglobulin); 2, 3 and 4 Protein
samples
Figure 12.1 Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of proteins.
Buffers
(i) 10% Ammonium persulphate
Ammonium persulphate – 0.1 g
dd.H2O – 1 mL
Prepare the solution immediately prior to use in 1.5 mL microfuge tubes. Make a fresh
each day.
(ii) Fixing solution
CH3OH – 50%
CH3COOH – 10%
ddH2O – 40%
(iii) Storing solution
CH3COOH – 7%
(iv) Staining guidelines: Place the gel in a box and cover it with fixing solution and fix
for 2 hours or O/N.
Pour out fixing solution and pour staining solution and agitate slowly for 4 hours.
Pour out staining solution and rinse with fixing solution and pour the destaining solution
I for 1 hour and destaining solution II still the bands are clear. Store the gel in 7%
CH3COOH.
Stock solutions to be prepared: All solutions must be filtered before use.
%$ Laboratory Manual for Genetic Engineering
(v) Acrylamide: Bisacrylamide (Acrylamide – 30%: Bisacrylamide – 2.67%)

Acrylamide – 58.4 g
N’-N’ Bismethylene acrylamide – 1.6 g
ddH2O upto 200 mL
(Filter and store at 4°C in the dark or brown bottle or wrap the bottle with tin foils and
store up to 30 days).
(vi) Resolving gel buffer (1.5 M Tris-HCl pH 8.8)
Trizma base – 18.15 g
ddH2O up to – 100 mL
Adjust the pH 8.8 with conc. HCl before making up to 100 mL and store at 4°C.
(vii) Stacking gel buffer (0.05 M Tris-HCl pH 6.8)
Trizma base – 6.0 g
Adjust the pH 6.8 with conc. HCl before making up to 100 mL and store at 4°C.
(viii) Tank buffer stock (10%)
Trizma base – 15 mL }
10% SDS – 100 mL Make up to 500 mL with dd H2O and before
use dilute 1:9 with water and store at 4°C
Glycine – 72 g
(ix) Stain stock (1% Coomassie Brilliant Blue R-250)
Coomassie Brilliant Blue R-250 – 2.0 g
ddH2O up to – 200 mL }
Stir well and filter
(x) Stain (0.125% CBB-R250; 50% methanol; 10% acetic acid)

Coomassie Brilliant Blue R-250 (from stock) – 62.5 mL
Methanol – 250.0 mL
Acetic acid – 50.0 mL
(xi) SDS-Sample buffer 2X for 10 mL
Stacking gel buffer (pH 6.8) – 2.5 mL
U|
10% SDS – 4.0 mL
|V
||
b mercaptoethanol – 1.0 mL Make up to 10 mL with ddH2O
Glycerol – 2.0 mL
Bromophenol Blue (0.15%) – 1.0 mL W
(1.5 mg in 1 mL of ddH2O)
(xii) Renaturing buffer 50 mL
4 M Urea – 12.012 g
50 mM NaCl – 0.1461 g
2 mM EDTA – 500 ml of 0.2 M EDTA
10 mM Tris-Cl (pH 7.0) – 0.0605 g
0.1 mM DTT – 10 ml of 0.5 M DTT
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Proteins %%
(xiii) Transfer buffer 2 litres

50 mM NaCl – 5.884 g
2 mM EDTA – 20 mL of 0.2 M EDTA
10 mM Tris – 2.422 g
0.1 mM DTT – (Adjust the pH 7.0 with HCl)
(xiv) 10X Towbin buffer 1 litre
25 mM Tris – 30.3 g
192 M glycine – 144 g
10% SDS – 100 mL
20% Methanol – 200 mL
Adjust volume to – 1 L ddH2O
(xv) CAPS buffer
10 mM CAPS buffer 1.11g/500 mL pH to 11.0
12.2 SILVER STAINING OF PROTEIN GELS

Silver nitrate staining of proteins after SDS-PAGE is quicker and ~10 times more sensitive than
Coomassie brilliant blue staining procedures and hence, nowadays widely used in research
laboratories.
Principle
The ability of silver to develop images was discovered in mid 17th century and subsequently
utilized in histochemical staining procedures. Silver nitrate staining of proteins essentially depends
on the reduction of silver ions to its metallic form. It has been proposed that the silver ions in
alkaline conditions complexes with proteins through e-amino group of lysine and sulphur group
of cysteine and methionine residues. When the complexed silver ions are reduced in the presence
of formaldehyde, they become metallic silver, which is seen on the gel as bands. Any free silver
ions must be washed-off the gel prior to development, as precipitation of silver oxide will result
in high background.
Protocol
1. After electrophoresis, transfer the gel to the fixing solution – I and incubate for
30 minutes (The gel can be stored for longer time in this solution) followed by fixing
solution – II for 10 minutes.
2. Wash the gel twice with ~ 50 mL water for 5 minutes each time to remove excess
methanol and formaldehyde.
3. Soak the gel in 0.02% thiosulphate solution for two minutes and rinse with water.
4. Incubate the gel in silver nitrate solution for ~ 30 minutes.
5. Rinse the gel with water twice (for 5 minutes each time) to remove excess silver ions.
6. Incubate the gel in 100 mL of the developing solution until bands start appearing.
7. As soon as the bands are seen, decant the developing solution and stop the reaction
immediately by adding 10 mL of the citric acid stop solution.
%& Laboratory Manual for Genetic Engineering
8. Store the gel in 10% acetic acid methanol solution (For long term storage 10% glycerol
may be included in the strong solution).
Buffers
(Use deionized double distilled water for preparation of solutions and for washing procedures).
(i) Fixing solution – I
Water – 50 mL
Methanol – 50 mL
(ii) Fixing solution – II
Water – 50 mL
Methanol – 35 mL
37% formaldehyde – 15 mL
(iii) 0.02% Sodium thiosulphate solution
Sodium thiosulphate – 20 mg
Water – 100 mL
(iv) 0.1% Staining solution
Silver nitrate – 100 mg
Water – 100 mL
(v) Developing solution
K2CO3 or Na2CO3 – 3 g
Water – 100 mL
0.02% thiosulphate – 3 mL
Solution
37% formaldehyde – 60 mL
(vi) Stopping solution
Citric acid – 5 g
Water – 100 mL
(vii) Gel storing solution
Glacial acetic acid – 10 mL
Methanol – 10 mL
Water – 80 mL
(for staining of mini gels, the volume of the above solutions can be reduced to half).
REFERENCES
Laemmli, U.K. (1970), Cleavage of structural proteins during the assembly of the head of
bacteriophage T4, Nature, 227 (5259), pp. 680–685.
Morrissey (1981), A Modified Silver Stain for Proteins, Anal. Biochem, 117, pp. 307–310.
b -Galactosidase Assay 79
13
b -Galactosidase Assay
The source of b-galactosidase enzyme is the bacterium, Escherichia coli, although

b-galactosidase is also found in many other micro-organisms, plants and animals, including
humans. b-galactosidase catalyzes the breakdown of the substrate lactose (the major sugar
present in milk) to two products, galactose and glucose compounds which is readily fed into the
glycolytic pathway:
b-galactosidase
Lactose Galactose + Glucose
All of the enzymes and other proteins in E. coli may be divided into two groups based on
whether their production is regulated or not:
1. Constitutively expressed enzymes are those enzymes that are continuously synthesized
in the cell (e.g. the enzymes of glycolysis or the lac repressor protein);
2. Inducible enzymes are those enzymes that are synthesized in the cell only when an
inducer (signal) is present in the cell (e.g. b-galactosidase or the enzymes in the
tryptophan biosynthesis pathway).
This procedure is designed to induce and measure the level of b-galactosidase in E. coli. Cells
grown in the absence of lactose do not synthesize b-galactosidase. If these cells are placed in
a medium containing lactose, b-galactosidase is produced within minutes, enabling the cells to
use lactose as a source of carbon and energy for growth. Some compounds such as, lactose,
isopropyl-b-thiogalactoside (IPTG), phenyl-b-galactoside (PBG) and glucose (GLU) are the
inducers of the synthesis of b-galactosidase and some compounds are the substrates for the
activity of b-galactosidase.
Enzyme assay
As already noted, the normal biological substrate for b-galactosidase is lactose. In theory, one
can measure enzyme activity by determining the rate of disappearance of the substrate (lactose)
or the rate of formation of a product (galactose or glucose). In assaying for b-galactosidase
activity, there are two difficulties with either of these methods:
79
1. There is no simple, rapid and inexpensive way to quantify lactose, galactose or glucose;
and
2. When one is using a crude cell extract as a source of b-galactosidase, there are many
other enzymes present that will immediately convert any galactose or glucose formed to
other compounds.
Ortho-nitrophenyl-b b -galactoside (ONPG): In the presence of b-galactosidase ONPG is
converted to galactose and ortho-nitrophenyl (ONP). E. coli cells contain no enzymes capable
of degrading ONP further. ONPG is colourless. ONP is also colourless at neutral or acid pH, but
in an alkaline solution, it is bright yellow. The amount of yellow colour can be measured in a
spectrophotometer and can be used as a measure of the amount of ONP formed in a given time.
Since ONP is a product of b-galactosidase activity, the spectrophotometric measurements can
be used as a reliable assay method for the enzyme.
Protocol
A. Growth of starved E. coli:
(a) Inoculate cells (E. coli strain CSH-141, a lac + strain) into 5 mL basic medium plus
2% glycerol and shake overnight at 37°C.
(b) Approximately 2 hours before use add 2.5 mL of O/N culture to 50 mL basic
medium plus 2% glycerol. Cells that are in log phase and starved should be used
for the assay. Glycerol is not a good energy source, so the cells are not able to grow
fast. By diluting the overnight culture and letting it grow for two hours and then
the cells will enter the log phase of growth.
B. Induction of enzyme: The synthesis of b-galactosidase may be induced using the
following procedure. Into a large size (18 mm) labelled test tubes add the following:
(a) 4 mL of starved E. coli cells (at a density of 1 ¥ 107 cells/mL).
(b) 0.2 mL of .002 M inducer (LAC, GLU, IPTG, PBG, or dH2O).
Put a cap on each tube, place in a 37°C water bath and aerate (shake) for 30 minutes.
C. Assay for enzyme: Although ONPG is used to determine whether or not b-galactosi-
dase has been synthesized in the cell, the compound will not quickly pass through a living
cell membrane. Therefore, the E. coli must first be treated with a detergent, sodium
deoxycholate, and an organic solvent, toluene, to destroy the selective permeability of
the cell membrane. This treatment, which allows ONPG to enter the cell quickly, also
kills the cells, but does not affect the activity of the enzyme (These compounds are also
toxic to humans and hence must be handled with caution).
13.1 DISRUPTION OF SELECTIVE PERMEABILITY

To 4.2 mL of an induced E. coli culture add:
1. One drop of sodium deoxycholate (from 1.0 mg/mL stock).
2. One drop of toluene.
Cap the tube and place in a 37°C water bath and aerate or shake for 10 minutes. This
preparation may be used for enzyme assays. Keep it in an ice bucket until use.
b -Galactosidase Assay &
13.2 ENZYME ASSAY

Into each small labelled culture tubes add:
1. 2.0 mL of 0.1 M sodium phosphate buffer (pH 7).
2. 2.0 mL lysed E. coli preparation.
3. 0.2 mL of 0.01 M ONPG (substrate).
Incubate for 15 minutes at 37°C without shaking. Stop the reaction by adding 0.5 mL 2 M
sodium carbonate. This will make the solution alkaline (pH > 8) and denature the enzyme. Read
the absorbance at 420 nm in a spectrophotometer. If the compound is an inducer, more enzymes
will be formed, more substrate (ONPG) will be converted to a yellow product and the absorbance
will be higher.
REFERENCE
MacGregor, G.R., G.P. Nolan, S. Fiering, M. Roederer and L.A. Herzenberg (1991), Use of
E. coli lacZ (b-Galactosidase) as a Reporter Gene, Methods in Molecular Biology, 7,
pp. 217–235.
14
Transduction of Plasmid DNA Using
CP-51 and CP-54 Bacteriophages
Transduction is the process by which genes are transferred from one bacterium to another
bacterium by bacteriophages (virus that infect bacteria). Transduction is a common tool used by
molecular biologists to stably introduce a foreign gene into a host cell’s genome.
When bacteriophages infect a bacterial cell, their normal mode of reproduction is to harness
the replicational, transcriptional, and translational machinery of the host bacterial cell to make
numerous virions, or complete the replication of viral particles, including the viral DNA or RNA
and the protein coat. However, the packaging of bacteriophage DNA has low fidelity and small
pieces of bacterial DNA, together with the bacteriophage genome, might be packaged into the
bacteriophage genome. At the same time, some phage genes are left behind in the bacterial
chromosome. When the bacteriophage progenies thus formed, infect fresh bacterial cells, deliver
their genome along with the extra-packaged host gene and the gene now has a chance of
integrating to the host cell chromosome and these genes are said to be transduced.
14.1 TRANSDUCTION OF PLASMID IN BACILLUS SP. WITH

CP-51 AND CP-54 PHAGE
The following are the methods for working with CP-51 and CP-54:
Recovery of phage from infected spores

(i) Inoculate 0.1 mL of infected spores into 25 mL of NBY broth containing 0.4% (W/N)
glycerol (added aseptically). Use a cotton plugged 250 mL flask. Incubate on shaker at
37°C for about 16 hours. Centrifuge for 15 minutes at 10,000 rpm in an SS-34 rotor
centrifuge. Be sure to do the centrifugation and all other operations at room temperature
or below 15°C. Filter through DA Millipore membrane.
(ii) If your source of phage is filter paper discs impregnated with phage-infected spores,
substitute such a disc for 0.1 mL of infected spores.
82
Transduction of Plasmid DNA Using CP-51 and CP-54 Bacteriophages &!
Assay of CP-51 and CP-54

(i) Use PA agar plates at room temperature overnight. For overlay onto 2 mL of soft PA agar
(0.05%).
(ii) To 2 mL of soft agar add 0.1 N of phage dilution (in 1% peptone) and approximately
2 ¥ 107 spores of B. cereus 569.
(iii) Incubate plates at 30°C or 37°C for 16–20 hours. There would be a loss of moisture
problem at 30°C.
Propagation of CP-51 and CP-54

(i) Pick 5 plaques (choose the most turbid ones) from a fresh assay plate (569 indicated)
and suspend in 5 mL of PA broth.
(ii) Mix on a vortex mixer.
(iii) If a host other than W/T B. cereus 569 is to be used for propagation, filter the phage
suspension through a Millipore HA or DA membrane.
(iv) To 3 mL of soft PA agar add 0.5 mL of phage suspension and 0.5 mL of cells or spores
(for B. anthracis use 1 mL of cells).
(v) Plate onto freshly prepared NBY agar plates containing 0.5% (v/v) of glycerol.
(vi) Incubate plate at 37°C for 18 to 20 hours.
(vii) Cells should be used as seed for the propagation of CP-51 to be used for plasmid
transduction if the plasmid contains an antibiotic resistance marker.
(viii) The inoculation for the broth culture of cells should be a colony from a selective plate.
(ix) Grow cells at 37°C for 18 to 20 hours in Luria broth with 0.4% glycerol or BHI broth
with 0.4% glycerol.
(x) Harvest each plate in 5 mL of PA broth. Centrifuge at 15°C to pellet the cells and filter
through DA Millipore membrane.
(xi) The yield is usually greater than 10 PFU per mL (sometimes the yield is less when
B. anthracis is the host).
(xii) Dilute 1 to 10 in PA broth having 0.02 M MgSO4 added aseptically along with 10% (w/
v) of DMSO and store at 15°C.
Plasmid transduction of antibiotic resistance with CP-51

Grow recipients in BHI broth containing 0.4% glycerol (w/v) overnight (16 to 18 hours).
Transfer 2.5 mL to 25 mL of BHI broth containing 0.4% glycerol in a 250 mL flask and incubate
on shaker for 6 to 7 hours.
(i) B. anthracis: Spread together 0.1 mL of phage lysate and 0.1 mL of recipient cells on
an HA or DA multipore membrane placed on an L agar plate. Incubate for 4 hours at
37°C to allow phenotypic expression of plasmid encoded antibiotic resistance. Then
transfer the membrane to L-agar containing a selective concentration of antibiotics and
incubate on additional 30 hours.
(ii) B. cereus and B. thuringiensis: Use 1-2 mL of recipient cells culture containing
4 ¥ 108 to 8 ¥ 108 cells per mL, mixed in a 20 mm tube with 0.5 to 1 mL of phage
suspension. Incubate on a shaker at 37°C. After 1 hour, 0.1 mL of phage antiserum
(diluted 1:10) is added, and continue incubation for an additional 1–2 hours to allow
&" Laboratory Manual for Genetic Engineering
phenotypic expression of the antibiotic resistance. Samples of 0.1 mL are spread

together with 0.1 mL of phage antiserum (diluted 1:10) on L-agar supplemented with
appropriate antibiotics. Plates are to be incubated at 37°C, and transductants are scored
after 36 hours.
Protocol
1. Inoculate CP-51 (in BC 569) in 3 mL of NBY with 0.4% glycerol.
2. Grow it for 36 hours at 30°C.
3. Centrifuge the culture at 15,000 rpm for 15 minutes. Take the supernatant.
4. Grow B. thuringiensis subsp. kurstaki 73.26 (pBC16) in NBY with 0.4% glycerol
overnight with 5 mg/mL tetracycline.
5. Subculture the overnight culture into a fresh NBY with glycerol and tetracycline, grow
it for 4 to 5 hours at 30°C.
6. Take 100 mL of phage supernatant and mix with 100 mL of B. thuringiensis subsp.
kurstaki 73.26 (pBC16) cultures. Add 2 mL of soft agar (42°C) and pour it on PA plate.
Incubate at 30°C for 12 hours.
7. Incubate Bacillus thuringiensis var. isralensis (Bti) in 3 mL of NBY with 0.4% glycerol.
8. Grow it for 36 hours at 30°C.
9. Subculture the overnight culture into a fresh NBY with glycerol for 4 to 5 hours at 30°C.
10. Add 5 to 6 mL of 1% peptone scrap off the 0.5% agar containing plaques. Spin for
15 minutes and 15,000 rpm. Pass the supernatant through a sterile Millipore membrane
and use the filtrate.
11. Mix 0.2 mL of the phage lysate (filtrate) with 2 mL of Bti (log phase culture).
12. Incubate at 37°C for 1 hour.
13. Spin down and wash the pellet twice with Luria broth and resuspended the washed cells
in 1 mL of fresh Luria broth and allow for expression for 1 hour at 37°C in slow shaking.
14. After 1 hour, plate the cells on suitable selective medium.
15. Incubate the plates at 30°C for 20 hours and score the transductants.
Buffers
(i) NBY broth (1000 mL)
Difco nutrient broth – 8.0 g
Difco yeast extract – 3.0 g
Distilled water upto – 1000 mL
(ii) PA broth (phage assay) (1000 mL) (pH 5.9 – 6.0)
Difco nutrient broth – 8.0 g
NaCl – 5.0 g
MgSO4 ◊ 7H2O – 0.2 g
MnSO4 ◊ H2O – 0.05 g
CaCl2 – 2.15 g
Distilled water upto – 1000 mL
Transduction of Plasmid DNA Using CP-51 and CP-54 Bacteriophages &#
(iii) Peptone
1% w/v Difco peptone
(iv) TBAB
Difco tryptone blood agar base
(v) TBAB mix
Difco tryptone – 66.0 g
Difco TBAB – 11.0 g
(tryptose blood agar base)
(vi) Nutrient broth (1000 mL) (pH 7.4 ± 0.2)
Peptone – 5.0 g
NaCl – 5.0 g
Beef extract – 1.5 g
REFERENCES
Canosi, U., G. Luder and T.A. Trautner (1982), SPP1-Mediated Plasmid Transduction, J. Virol,
44(2), pp. 431–436.
Deichelbohrer, I., J.C. Alonso, G. Lüder and T.A. Trautner (1985), Plasmid transduction by
Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and
bacteriophage, J. Bacteriol, 162 (3), pp. 1238–1243.
&$ Laboratory Manual for Genetic Engineering
15
Bacterial Conjugation
Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-
cell contact. Joshua Lederberg and Edward Tatum (1946), discovered bacterial conjugation as a
mechanism of horizontal gene transfer. Bacterial conjugation is often incorrectly regarded as the
bacterial equivalent of sexual reproduction or mating. It is not actually sexual, as it does not involve
the fusing of gametes and the creation of a zygote, nor is there equal exchange of genetic material.
It is merely the transfer of genetic information from a donor cell to a recipient cell. In order to
perform bacterial conjugation, one of the bacteria, the donor, must play as a host to a conjugative
or mobilizable genetic element, most often a conjugative plasmid. Most conjugative plasmids have
genes to ensure that the recipient cell does not already contain a similar genetic element.
The genetic information transferred is often beneficial to the recipient cell. Benefits may
include antibiotic resistance, other xenobiotic tolerance, or the ability to utilize a new metabolite.
Bacterial conjugation is considered as a mechanism which was evolved by the mobile genetic
element, to spread itself into new hosts.
15.1 CONJUGAL TRANSFER OF DNA INTO CYANOBACTERIA

Triparental mating
Three parental strains used for triparental mating are given below:
1. E. coli having the conjugal plasmid (e.g. RP4).
2. E. coli having the cargo plasmid + helpers in DH5a.
3. The target Cyanobacterium (e.g. Anabaena sp.).
Log phase cultures from single colonies of the three parental strains are brought together on
a nitrocellulose filter disc resting on solid cyanobacterial medium. After a day on non-selective
medium, the filter is transferred to a selective cyanobacterial medium. It is sometimes necessary
to use different dilutions of cyanobacteria in the mating experiments. Non-mated cyanobacteria
may grow despite selection against them.
Two types of mating can be done such as spot mating and plate mating.
The spot mating is useful in testing a new strain or a new plasmid and plate mating is useful
in finding rare transconjugant.
86
Bacterial Conjugation &%
Protocol
Preparation of E. coli
1. Grow each E. coli strain mentioned above in Luria broth plus appropriate antibiotics.
2. Harvest the E. coli cells in late exponential phase (sometimes overnight cultures appear
to work as well). Antibiotic concentrations generally used for E. coli are as follows:
Ampicillin 50 mg/mL; Kanamycin 50 mg/mL; Tetracyclin 15 mg/mL; Chloramphenicol
25 mg/mL; Streptomycin 25 mg/mL.
3. For plate matings, cargo strains (10 mL/plate of each) and a conjugal transfer strain
(volume equal to sum of cargo volumes) are needed.
4. For spot matings cargo strain, anything greater than 0.75 mL (each) and a conjugal
strain (0.75 mL per cargo strain) are needed.
5. Mix three parental strains with appropriate concentrations and add to a sterile filter kept
over cyanobacterial non-selective medium (BG 11). Incubate for 24 hours and transfer
the nitrocellulose filter paper disc to a selective cyanobacterial medium (BG 11 with
respective antibiotics) and incubate in dim light (25°C) till isolated cyanobacterial
colonies appear on filter paper disc.
6. Individually culture the isolated colonies and screen for the presence of the plasmid and
the expression of the gene present in it.
15.2 CONJUGAL PLASMID TRANSFER IN B. THURINGIENSIS

Protocol
1. Maintain strains of B. thuringiensis on slants of nutrient salts agar and grow in nutrient
broth.
2. Cry+ donor strain (antibiotic sensitive) and Cry– recipient strain (antibiotic resistant) grow
separately in nutrient broth in a shaker incubator for 14 to 20 hours for overnight at 30°C.
3. These stationary phase cultures are to be diluted 1:50 into fresh nutrient broth and
incubated with shaking for 3 to 4 hours.
4. Perform mating in nutrient broth by mixing 10 mL/mL of donor and recipient cultures
usually in a final volume of 2 mL for 8 to 20 hours.
5. The recipient population is to be selected by streaking on to antibiotics supplemented
nutrient salts agar.
6. At the end of mating experiments, samples are to be serially diluted and streaked on to
antibiotic supplemented nutrient salt plates and incubated at 30°C for 48 hours.
7. Usually Cry+ colonies are whiter and glossy than adjacent acrystalliferous colonies.
Buffer
(i) Nutrient salt agar (1000 mL) (pH –7.0)
Nutrient broth – 8.0 g
Bacto agar – 15.0 g
CaCl2 – 0.1 g
MnCl2 ◊ 4H2O – 10.0 g
&& Laboratory Manual for Genetic Engineering
MgCl2 ◊ 4H2O – 0.2 g

15.3 INTRODUCTION OF BINARY PLASMIDS INTO

AGROBACTERIUM BY TRIPARENTAL MATING
Agrobacterium tumefaciens LBA 4404 harbours a derivative of Ti-plasmid that lacks the T-DNA
region but contains the vir region (vir helper strain). This strain is grown at 30°C on AB minimal
medium + Rifampicin 10 mg/mL. Binary plasmid pGA472 in E. coli contains the right and left
sequences of T-DNA boarder. This plasmid multiplies in E. coli and in Agrobacterium. E. coli
harbouring this plasmid grows on Luria broth with tetracycline 20 mg/mL. Agrobacterium with
this plasmid also grows on AB minimal medium with tetracycline 5 mg/mL. Plasmid pGA 472 is
not self-transmissible from E. coli to Agrobacterium. Transfer functions for conjugal transfer
are supplied by introducing another conjugative plasmid pRK2013 in E. coli. This plasmid, when
introduced into E. coli strain harbouring pGA472, mobilizes pGA472 into Agrobacterum. E. coli
harbouring pRK2013 grow on Luria broth medium with 50 mg/mL Kanamycin. This plasmid can
also replicate in Agrobacterium.
Protocol
∑ The day on which triparental mating is performed is taken as day 1.
∑ A. tumefaciens LBA 4404 (vir helper strain is streaked to get single colonies on AB
minimal medium agar plates containing 10 mg/mL rifamycin. Incubate at 30°C.
Day 1:
∑ E.coli harbouring pRK2013 is streaked to single colonies on Luria agar medium with
50 mg/mL Kan. E. coli harbouring pGA472 is streaked to single colonies on LB minimal
medium with 20 mg/mL tet. Grow at 30∞C.
An overnight culture is grown from each strain by inoculating single colony and grown at
30∞C with suitable antibiotics on broth (YEP). Sub-culture the overnight culture on broth with
suitable antibiotics. Prepare a plain YEP agar plate and place a nitrocellulose filter paper disc at
the centre. Mix 25 mL from each culture in a sterile tube and put on the filter and let it air dry
in a laminar flow hood. After all the broth is absorbed or dried up, the plate should be incubated
at 30∞C for 24 hours.
Day 2:
∑ 4 plates with minimal medium supplemented with rif r tet r are to be prepared. The mixed
culture in the filter is to be taken out and resuspended in 0.9 mL of 0.9% NaCl. Vortex
well and serially dilute and plate on AB minimal medium plates supplemented with ref r
tetr. The plates are to be incubated for 3 to 5 days.
Day 6:
∑ At one or two dilutions single colonies would appear on AB minimal agar plates. Those
colonies of A. tumefaciens LBA 4404 would grow only when PGA 472 has been
transferred, E. coli strains used in this experiment are auxotrophic mutants. Therefore,
they would not grow on AB minimal medium.
Bacterial Conjugation &'
∑ A. tumefaciens LBA 4404 lack tet resistance. Therefore, it would grow only if it acquires
pGA472 that carries a tet resistance gene.
Day 7:
∑ Six to eight single colonies of A. tumefaciens should be patched on AB minimal medium
plates with antibiotics. To confirm the plasmid transfer, screen the colonies by plasmid
extraction, slot lysis electrophoresis or southern hybridization.
Media and buffers

(i) AB minimal medium
Liquid medium (100 mL)
Add 0.5 g of glucose in 90 mL of water. Autoclave and cool. To this add 5 mL of
20X AB buffer and 5 mL of 20X AB salts.
(ii) Solid medium: 0.5 g of glucose and 1.5 g of agar added to 90 mL of water. Autoclave
and cool to about 55°C. To this add 5 mL of 20X AB buffer and 5 mL of 20X AB salts.
(iii) AB salts (20X) 100 mL
Ammonium chloride – 2.0 g
Magnesium sulphate 7H2O – 0.6 g
Potassium chloride – 0.3 g
Calcium chloride 2H2O – 0.3 g
Ferrous sulphate 7H2O – 5 mg
Volume made upto 100 mL. No need to adjust the pH. Autoclave and store.
(iv) AB buffer (20X) 100 mL (pH 7.0)
K2HPO4 – 6.0 g
NaH2PO4 – 2.0 g
Make up to 100 mL, autoclave and store.
(v) YEP medium/litre (pH 7.2)
Yeast extract – 10 g
NaCl – 5g
Peptone – 10 g
REFERENCES
Chapman, J.S. and B.C. Carlton (1985), Conjugal plasmid transfer in Bacillus thuringiensis, Basic
Life Sci, 30, pp. 453–467.
Charles, H. Shaw (1995), Plant Gene Transfer and Expression Protocols, pp. 33–37.
Elhai, J. and C.P. Wolk (1988), Conjugal transfer of DNA to cyanobacteria, Methods Enzymol,
167, pp. 747–754.
Lederberg, J. and E.L. Tatum (1946), Gene recombination in Escherichia coli, Nature, 58: 558.
' Laboratory Manual for Genetic Engineering
16
Blotting Techniques
The Blotting techniques are used for transferring the macromolecules, such as nucleic acids and
proteins from the gel on to either nitrocellulose membrane or nylon membrane. The large-sized
nucleic acid molecules need depurination before transferring on to membranes. The nitrocellulose
or nylon membranes after the blotting can be processed for hybridization with a specific probe,
either labelled with radioactive isotope or with a chromogenic substance for subsequent
identification of the homologous macromolecule present in the original gel used for blotting.
There are three types of blotting techniques used in genetic engineering, viz., western blotting
(for proteins), southern blotting (for DNA) and northern blotting (for RNA).
16.1 WESTERN BLOTTING

Diffusion transfer
1. After electrophoresis of proteins in SDS-PAGE, immerse the gel in 200 mL of urea
containing buffer and gently agitate for 1 to 3 hours.
2. Then the gel is to be sandwiched between two strips of nitrocellulose membrane cut
exactly at the size of the gel and press it together in a sandwich apparatus.
3. Submerge the apparatus in a solution of transfer buffer (2 litres) for 36 to 48 hours.
Replace the initial solution after 12 hours.
4. All steps are to be carried out at room temperature and the protein is transferred by
diffusion.
Electrophoretic transfer using semiphor blotter unit

Keep one stained protein gel for reference and use another similar gel for blotting. Transfer the
proteins as soon as possible after electrophoresis to avoid diffusion of the proteins in the gel. Gels
transferred simultaneously should be of the same size.
Use buffer with low ionic strength to prevent overheating. It is found that both Towbin
buffer with SDS and 10 mM CAPS buffer provide the most efficient protein transfers under most
conditions.
90
Blotting Techniques '
Protocol
1. Remove the stacking gel from the gels and measure one of the gels and record the size.
2. Six pieces of blotter paper (Whatman No. 1) shaped exactly to the size of the gel, plus
one piece for each gel to be transferred are needed. It is important that the filter paper,
as well as the membrane, should be of the size of the gel. Larger pieces will make contact
around the gel and thereby allow the current to go around the gel, making transfer
inefficient.
3. Cut the membrane to the size of the gel. Keep the membrane in the transfer buffer for
2 to 5 minutes.
4. The opening in the Mylar mask should be about 2 mm smaller than the size of the gel
in both length and width. Cut an opening in Mylar mask or use a precut mask with an
opening approximate the size.
5. If the cover is still in place on the base, unplug the safety-in lead connecting the two and
lift the cover off.
6. Place the Mylar mask in the bottom of the semiphore, centring the open side over the
electrode.
7. In a dish of transfer buffer, saturate three pieces of filter paper cut large enough to
overlap. The cut-out mask in the Mylar mask, should not be larger than the gel. Place
these on the top of Mylar mask, centring them by placing the centre of the filter paper
down first, and then roll the edges out. The filter paper should cover the cut-out in the
mask and slightly overlay it on all sides.
8. Construct the first transfer sandwich on top of the blotter paper also in the semiphor by
placing the membrane then the gel, then more saturated filter (blotter) paper on the stack.
9. Add up to five additional transfer sandwiches on top of the first.
10. Place three pieces of buffer-soaked blotter paper on top of the entire stack.
11. The cover fits only one way. Hold the cover by the two handles. Align the three ridges,
one on each of three sides on the grooves in the bottom half of the semiphor. Hold the
cover level and slide it down gently onto stack. Do not remove the cover until blotting
is over.
12. Plug the short safety interlock-lead, which is attached to the cover, into the jack in the
base.
13. Occasionally, when transferring multiple protein gels, the lid is to be weighed in order
to ensure even contact within the stack of the gels and membrane. In the case, place
up to one kg weight on cover (Figure 16.1). Too much weight will compress the stack
and hinder transfer.
Transfer
1. Check that power supply is turned off. Connect the two larger leads to the power supply,
plugging the red lead into the positive jack and black lead into the negative jack.
2. Turn power supply to zero before switching it on. Do not use the semiphor with current
over 250 mA. For transfers exceeding two hours in duration, run the semiphor in a cold
room.
' Laboratory Manual for Genetic Engineering
Blotting paper
These stack components (2-3 sheets)
are the same size as the Gel
gel or slightly smaller
Membrane
Blotting paper
(2-3 sheets)
Mask (opening
approx. 2 mm
smaller than the
gel on all sides)
Figure 16.1 Transfer stack for a single gel.
3. Turn on the power supply and set it at approximately 0.8 mA/em2 of gel. A general rule
is that larger proteins, native proteins and their gels require longer transfer times. Use
Table 16.1 for guidelines.
4. Minigels of at least 8 ¥ 7 cm can be limited to 0.8 mA to avoid excessive heating during
transfer.
TABLE 16.1 Approximate duration of runs according to protein molecular weights*

Molecular weight in Dalton Transfer period
< 20,000 15 minutes
20,000 – 80,000 35 minutes
> 80,000 45 minutes
*Run at 100 volts on 8 ¥ 7 cm mini gels.
After transfer
1. After transfer, turn off the power supply. Disconnect the leads from the power supply
jacks.
2. Unplug the lead connecting the cover and base of the semiphor.
3. Remove the cover (Caution: part of the stack may stick to the cover).
4. If more than one gel was blotted, mark the gels and membranes for identification by
clipping the corners while removing that from the stack.
5. Process the blotted membranes for immunoblot analysis.
6. Rinse the semiphor thoroughly with distilled water and allow to air-dry at room tempera-
ture and store. Do not immerse the unit in water. The semiphor is not autoclavable.
Blotting Techniques '!
Note
While placing each layer on top of the stack, make sure that no air bubbles are trapped under-
neath. Sweep each layer with a gloved finger or roll a pipet or test tube over the layer to remove
bubbles. Placing a few drops of buffer on top of the area where a bubble is trapped, makes the
bubble easier to remove.
Buffers
(i) Towbin buffer
25 mM Tris
192 M glycine
0.1% SDS
20% Methanol
(ii) Urea buffer
8 M urea
2 M thiourea, 1% (w/v) CHAPS
20 mM DTT
0.8% (v/v) carrier ampholytes 3–10
100 mM Tris-HCl, pH 7.5
1 mM EDTA
14 mM PMSF
16.2 IMMUNOBLOTTING ASSAY

1. Nitrocellulose filters after protein transfer should be washed with 100% of TBS.
2. Then the membrane is to be immersed into blocking solution and is to be gently agitated
for 2 hours to 3 hours.
3. Then the membrane should be removed from blocking solution and is to be transferred
to a fresh tray containing 10 mL of first antibody buffer. First antibody is used at 1:1000
dilution and incubated for 2 to 4 hours or O/N at room temperature.
4. The membrane is then to be transferred from first antibody buffer and rinsed with water,
and again with TTBS for 20 minutes and with TBS for 10 minutes (two times).
5. Then transfer the membrane to second antibody buffer. Second antibody is used at a
1:2000 dilution. Incubate at room temperature for 1 hour.
6. Then the membrane is to be rinsed, washed once with TTBS for 10 minutes and then
with TBS for 10 minutes.
7. Then the membrane is to be immersed in 10 mL of freshly prepared HRP colour
development solution and kept in darkroom for 5 minutes.
8. Colour reaction will be seen in the corresponding protein bands within 5 minutes.
Buffers
(i) 1X TBS
Tris pH 7.5 – 10 mM
NaCl – 0.9%
'" Laboratory Manual for Genetic Engineering
(ii) Blocking solution

3% Gelatin in TBS or 1% BSA in TBS
(iii) Antibody buffer
1% Gelatin in TBS or 1% BSA in TBS
(iv) TTBS
0.05% Tween 20 in TBS
(v) HRP colour development solution
0.124 g colour development buffer in 10 mL d.H2O
100 mL colour development reagent NBT (nitro blue tetrazolium)
100 mL BCIP (5-Bromo-4-chloro-3-indolyl phosphate)
(NBT 3% in 70% DMF – 15 mg NBT in 500 mL of 70% DMF
BCIP 1.5% in DMF – 7.5 mg BCIP in 500 mL of DMF)
(vi) Alkaline phosphatase (AP) colour development buffer
Tris – 0.1 M
MgCl2 – 0.5 mM
}
pH 9.5
AP 0.1 mg/mL in 50% glycerol use at 1:2000 dilutions in TBS with 1% BSA.
16.3 SOUTHERN BLOTTING

16.3.1 Capillary Blotting or Passive Diffusion
Blotting on Nitrocellulose
Protocol
1. Run the agarose gel and stain it with ethidium bromide and take photograph of the gel
for reference.
2. Depurinate the DNA in the gel by immersing it in 0.25 mL of HCl for 1 hour.
3. Rinse the gel in ddH2O several times.
4. Soak the gel in 1 litre (1 M NaCl) and 0.5 M NaOH for 1½ hours.
5. Rinse the gel in ddH2O several times.
6. Soak the gel in 1 litre of 1.5 M NaCl, 0.5 M Tris pH 7.0 for 1½ hours for neutralization.
7. Now soak the gel in 20X SSC for 1 hour.
8. Float the nitrocellulose filter on the surface of 2X SSC until it wets completely from
beneath. Then immerse the filter in the 2X SSC for 2-3 minutes (Do not touch the
nitrocellulose with hands, use hand gloves).
9. Cut a stack of paper towels to stack up to 5.8 cm height.
10. Over this, keep 2 pieces of (Whatman 3 mm) paper cut exactly to the size as the gel,
in 2X SSC and place it over the paper towels.
11. Invert the gel and leave it gently over the nitrocellulose membrane kept above the
Whatman 3 mm (bottom nitrocellulose paper).
12. Place another nitrocellulose, cut exactly to the size of the gel, over the gel (moisten the
NC with 2X SSC before use).
13. Keep 2 Whatman 3 mm paper soaked in 2X SSC over this.
Blotting Techniques '#
14. Over this, keep a stack of paper towels up to 5 to 8 cm height.

15. Over this, keep a weight of 500 g and allow the transfer overnight or for 36 hours
(Figure 16.2).
Weight 0.5 kg
Glass plate
Stack of filter paper towels

3 sheets of Whatman no.3 filter paper
Nitrocellulose membrane
Agarose gel kept upside down
Filter paper wick
Tray containing salt solution
Figure 16.2 Set up of capillary blot.
16. Remove the towels and the 3 mm filter above the gel and below the gel.
17. Remove the nitrocellulose membrane filters and dry it on a 3 mm paper.
18. Mark the position of the gels slots on the filters with a very soft pencil or a ball point
pen.
19. Stain the gel and check for efficiency of transfer (There should not be any fluorescence
under UV).
20. After drying the filter at room temperature on a sheet of 3 mm paper, sandwich them
between two sheets of 2 mm paper and bake for 2 hours at 80°C under vacuum or fix
the DNA in the membrane by UV cross-linking.
21. Store between sheets of 3 mm paper at room temperature until filters are used for
southern hybridization.
Buffers
(i) Depurination solution
0.25 M HCl – 1 litre
(ii) Denaturation solution 1 litre
(1 M NaCl, 0.5 M NaOH)
NaCl – 58.44 g
NaOH – 20.0 g
(iii) Neutralizing buffer 1 litre
(1.5 M NaCl, 0.5 M Tris pH 7.0)
NaCl – 87.66 g
Tris – 60.5 g
(iv) 20X SSC 1 litre
NaCl – 175.3 g
Na citrate – 88.2 g
'$ Laboratory Manual for Genetic Engineering
800 mL water. Adjust the pH with 10 N NaOH to 7.0 and make up to 1000 mL and
autoclave.
(v) 2X SSC 100 mL
20X SSC – 10 mL
ddH2O – 90 mL
Prior to starting the experiments keep crude filter paper towels, Whatman 3 mm and
Nitrocellulose membrane ready. Also keep ready four trays (cleaned), 500 g weight and a big tray
in which the transfer could be carried out.
16.3.2 Southern Blotting using Semiphor Blotting Unit

The Transfer Method described here is a new, preliminary method, which provides general
guidelines for electrophoretically transferring DNA to nylon membrane charged or uncharged.
The electrophoresis buffer used to run the gel, whether TBE or TAE, can be diluted and used
as transfer buffer. The ionic strength of the buffer is 350–400 mM.
Gel treatment
Protocol
1. Run the agarose gel (up to 5 mm thick) under standard conditions.
2. Stain gel with Ethidium Bromide (Et.Br.) by soaking it in a solution of 0.5 mg/mL ethidium
bromide. Alternatively, the Et.Br. may be added directly to the gel running buffer.
3. Depurinate the gel by soaking it in depurination solution with gentle agitation for
15 minutes. (This is to increase the efficiency of transfer).
4. To denature the DNA in the gel prior to transfer, soak the gel in denaturation solution
with gentle agitation for 20 minutes. Neutralize the gel by soaking in neutralization
solution, 2 times for 15 minutes each.
5. If the transfer is being made to a nylon membrane, which is base resistant (Nylon 66
plus) you can denature the gel before the transfer or instead, base wash the membrane
following the transfer. If the nylon membrane being used is not base resistant, the DNA
should be denatured in the gel prior to transfer.
6. To base wash the membrane following transfer, soak the membrane in 0.1 N NaOH for
30 minutes. Neutralize the membrane by soaking in 0.2 M Tris-base (pH 8.0) with 0.5%
SDS for 30 minutes.
7. Cut 8 pieces of blotter paper and one piece of membrane to the size of the gel. Do not
cut the paper or membrane larger than the gel. If papers from either side of the gel stack,
touch each other during the transfer, current will travel through the paper around the gel.
8. Saturate the gel and the blotter papers in 0.1X TAE or 0.3X TBE (use the type of buffer
originally used to run the gel). Soak the membrane in ddH2O for 5 to 10 minutes and
then in 0.1X TAE or 0.3X TAE for 10 to 20 minutes. When measured with a conductivity
meter, the ionic strength of the diluted buffer should be 350–400 mmho.
9. Use a precut Mylar mask or cut a hole in the Mylar sheet slightly smaller than the size
of the gel by almost 2 mm on each side.
10. Stack the gel sandwich in the blotter unit as shown in Figure 16.3.
Blotting Techniques '%
Cover
(contains the cathode)
Safety-interlock Guides, (3)

lead
Mask
Safety-interlock
housing
Colour-coded
leads
Base
(contains the anode)
Figure 16.3 Blotter unit main components.
11. Place the Mylar mask on the anode (bottom) plate in the unit.
12. Stack 4 sheets of wet blotter paper over the hole in the Mylar mask.
13. Place the pre-soaked membrane on top of the blotter papers.
14. Centre the gel on top of the membrane.
15. Place 4 sheets of wet blotter paper over the gel.
16. Put the lid containing the cathode plate over the whole stack. Between each layer, roll
a pipette over the stack to remove any trapped air bubbles. This is especially important
on the layer between the membrane and the gel.
Note: When two or more nucleic acid gels are transferred at the same time, only the gel
at the bottom of the stack is transferred efficiently.
17. Plug the safety interlock lead from the lid into the base of the blotter unit and cover the
electrode to the power supply.
18. Transfer it a constant of 50 mAmp for 30 minutes.
19. After the transfer or after the blotting, process the membrane for southern hybridization.
Buffers
(i) 1X TBE (89 mM Tris, 89 mM Boric acid, 2.5 mM EDTA, pH 8.4)
Tris-base – 10.8 g
Boric acid – 5.5 g
Na2EDTA◊2H2O – 0.93 g
dH2O up to 1000 mL
'& Laboratory Manual for Genetic Engineering
(ii) 1X TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0)

Tris-base – 4.84 g
Glacial acetic acid – 1.14 mL
Na2EDTA◊2H2O – 0.37 g
adjust pH 8.0 with acetic acid and make up to 1000 mL
(iii) Depurination solution (0.25 M HCl)
HCl – 20.8 mL
dH2O – 1000 mL
(iv) Denaturation solution
NaOH – 20.0 g
NaCl – 58.4 g
dH2O up to1000 mL
(v) Neutralizing solution (0.5 M Tris-HCl, pH 7.4; 1.5 M NaCl)
Tris-HCl – 78.8 g
NaCl – 87.6 g
d.H2O up to 1000 mL
16.3.3 Colony Blotting

1. Prepare master plates of colonies to be hybridized and replica plate should also be
prepared for colony hybridization.
2. 4 pieces of (Whatman 3 mm) paper should be cut so that they could fit neatly on the
bottom of four plastic trays.
3. One piece of paper was saturated with 10% SDS, place the nitrocellulose membrane
filter on to the colonies on agar and presses it gently over the colonies. Using blunt-ended
forceps, the nitrocellulose filter was peeled off from the plate and was placed, colony
side up, on the SDS-impregnated 3 mm paper for 3 minutes.
4. Transfer the filter to the second sheet of 3 mm paper that had been saturated with
denaturing solution. Leave the filter for 5 minutes.
5. Neutralize the filter by placing colony side up on a 3 mm paper (Whatman), and add
neutralization solution and incubate it for 5 minutes.
6. Then put the filter in between 2 sheets of dry 3 mm paper and bake for 2 hours at 80°C
in a vacuum-oven.
Buffers
(i) 10% SDS
10 g in 100 mL of d.H2O
(ii) Denaturing solution for 250 mL
0.5 M NaOH – 5.0 g
1.5 M NaCl – 21.915 g
(iii) Neutralizing solution for 250 mL
1.5 M NaCl – 21.914 g
0.5 M Tris-HCl (pH 8.0) – 15.1375 g
Blotting Techniques ''
16.4 NORTHERN BLOTTING

1. After electrophoresis of RNA with formaldehyde containing gel is complete, soak the gel
for 5 minutes in several changes of water. (Gels containing formaldehyde are less rigid
than non-denaturing agarose gels. Care must be exercised in handling them).
2. Soak the gel in an excess of 50 mM NaOH and 10 mM NaCl for 45 minutes at room
temperature (The partial alkaline hydrolysis improves the transfer of high molecular
weight RNA).
3. Neutralize the gel by soaking for 45 minutes at room temperature in 0.1 M Tris-HCl
(pH 7.5).
4. Soak the gel for 1 hour in 20X SSC.
5. Transfer the RNA to nitrocellulose by the standard methods (either by capillary or
electrophoretic blotting).
6. After transfer is complete, wash the filter in 3X SSC, dry in air for 1–2 hours and bake
for 3 to 4 hours at 80°C under vacuum and process the filter for northern hybridization.
Buffers
Please refer to Section 16.2.
REFERENCES
Born, T.L. and C.G. Miyada (1991), Stained colonies facilitate alignment in non-radioactive
colony hybridization, Bio Techniques, 10 (4), pp. 480–481.
Lehrach, H., D. Diamond, J.M. Wozney and H. Boedtker (1977), Biochem, 16, pp. 47–43.
Neal Burnette, W. (1981), Western blotting: electrophoretic transfer of proteins from sodium
dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection
with antibody and radioiodinated protein A., Analytical Biochemistry, 112 (2), pp. 195–203.
Sambrook, J., and D.W. Russel (2001), Molecular Cloning: A Laboratory Manual, 3rd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Southern, E.M. (1975), Detection of specific sequences among DNA fragments separated by gel
electrophoresis, J Mol Biol, 98, pp. 503–517.
Towbin, H., T. Stachelin and J. Gordon (1979), Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications,” Proc
Natl Acad Sci USA, 76(9), pp. 4350–4.
17
32
P Labelled Probe Preparation and
Measurement of Radioactivity in
Radio-Labelled Nucleic Acid
Short oligonucleotides of random sequence can serve as a primer for the initiation of DNA
synthesis at multiple sites on single-stranded DNA templates. During extension of the primers by
DNA polymerase-I, the complement of every nucleotide in the template (except those at the
extreme 5¢ terminus) will be incorporated into the product at approximately equal frequency. The
DNA synthesized can be labelled by using one [-32P] dNTP and three unlabelled dNTPs as
precursors, generating probes with specific activities of 5 ¥ 108 to 5 ¥ 109 dpm/mg DNA.
Currently different probe preparation kits are marketed by molecular biology companies.
32
17.1 P LABELLED PROBE PREPARATION
17.1.1 Nick Translation or Oligolabelling with a 32P dCTP
Reaction mix
Denatured DNA (25–50 ng) – £ 34 mL
Reagent mix (provided) – 10 mL
(a32 P) dCTP (3600 Ci/mmol) – 5 mL (50 mCi)
dH2O up to – 49 mL
Klenow fragment – 1 mL
Total reaction volume = 50 mL
Mix and gently spin down and incubate at 37°C for 30–60 minutes.
Stop the reaction by adding 100 mL of dye mix. Separate the labelled probe from the
unincorporated DNA by gel-filtration through Sephadex G-50 column.
100
32
P Labelled Probe Preparation and Measurement of Radioactivity
17.1.2 Random Primer Labelling of Probe using a 32P dCTP

Reaction mix
1. DNA solution (25 ng) – 1-1 mL
2. Unlabelled dNTPs (dATPs
dTTPs, dGTPs) 4 mL each – 12 mL
3. Buffer (solution 1X) – 5 mL
4. Primer + BSA (Solution II) – 5 mL
5. a32 labelled dCTP – 5 mL
6. Water up to 48 mL
7. Enzyme – 2 mL
Total reaction volume 50 mL
Mix well and incubate at 37°C for 30 minutes to 1 hour stop the reaction by adding 100 mL
dye mix.
17.1.3 Separation of Probe from Unincorporated Label by

Gel-Filtration through Sephadex G-50 Column
Protocol
Preparation of Sephadex G-50: Slowly add 30 g of Sephadex G-50 to 250 mL of TE (pH 8.0)
in a 500 mL beaker or bottle. Make sure that the powder is well-dispersed. Let it stand over-night
at room temperature or heat at 65°C for 1–3 hours or autoclave for 15 minutes. Allow cooling
to room temperature. Decant the supernatant and replace with an equal volume of TE (pH 8.0)
and store at 4°C.
Column chromatography
1. Use a pasteur pipette, with a sterile glass wool plug, make a 2 mL Sephadex G-50 column.
2. Wash the column with several volume of column buffer.
3. Load sample onto the top of the column and wash the Eppendorf tube with 100 mL
column buffer and load the washings also on the top of the column.
4. Elute the labelled DNA with column buffer by collecting the blue fraction. Measure the
eluted volume and take 5 mL for counting radioactivity. Collect other different fractions
also.
17.2 MEASUREMENT OF RADIOACTIVITY IN

NUCLEIC ACID
17.2.1 Absorption to DE-81 Filter
1. Spot a known volume (up to 5 mL) on to the centre of each of two 2.4 cm disc of
Whatman DE-81 filter paper.
2. Wash one of the discs six times, 5 minutes per wash and twice in 0.5 M Na2HPO4. Then
wash the disc twice in water (1 minute per wash) 95% ethanol (2 minutes per wash).
3. Dry both filters and count in a liquid scintillation counter in an aqueous scintillation fluid.
17.2.2 Precipitation with Trichloroacetic Acid (TCA)

1. Spot the sample (upto 10 mL) at the centre of a Whatman (filter disc) glass fibre disc
(2-4 cm, diameter).
2. Add an equal volume of the sample to a tube containing 100 mL of solution of Salmon
sperm DNA (500 mg/mL in 20 mM EDTA). Add ice-cold 10% TCA, mix and chill on
ice for 15 minutes.
3. Collect the precipitate by filtering the solution through the Whatman filter disc or the
glass fibre disc. Wash the filter six times with 5 mL ice-cold 10% TCA followed by 5 mL
95% ethanol.
4. Dry the filters under a heat lamp. Put the filters into scintillation vial and count in a liquid
scintillation counter within a toluene based scintillation fluid. First filter measures the
total radioactivity in the sample. The second filter measures the radioactivity
incorporated into nucleic acids. Nucleic acids greater than the nucleotides in length are
quantitatively precipitated by this procedure.
Buffers
(i) Nick translation dye mix
Blue dextran – 6 mg/mL
Orange G – 1 mg/mL
EDTA solution (pH 8.0) – 0.05 M
(ii) Column buffer
Trizma base – 10 mM
EDTA – 1 mM
NaCl – 100 mM
(iii) Scintillation fluid
0.5% 2, 5, Diphenyl Oxazole in Toluene.
(iv) 20 mM EDTA
REFERENCE
Sambrook, J. and D.W. Russel (2001), Molecular Cloning: A Laboratory Manual,
3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Hybridization Techniques !
18
Hybridization Techniques
Hybridization is the technique in which the nucleic acid or protein immobilized on the membrane
is annealed with a homologous DNA/RNA. Hybridization is widely used to confirm the presence
or absence of the DNA/RNA in the unknown sample. Hybridization depends on the function of
the labelled base pair between the probe and the target sequence. After the completion of blotting
technique, the membrane is placed in a solution of labelled (radioactive or non-radioactive) single
stranded DNA or RNA solution. This DNA or RNA contains sequences complementary to DNA
or RNA present on the membrane. This labelled nucleic acid used to detect or locate homologous
DNA is called as probe. Conditions are chosen such that labelled DNA or RNA bind together or
hybridize with nucleic acid present on the membrane. If the sequence of nucleic acid in the probe
is complementary to nucleotide sequence on the membrane then base pairing or hybridization will
occur. The location of the hybridized probe can be subsequently detected by either autoradio-
graphy or by phosphor imager or by chemiluminescence and the homologous DNA or RNA; band
could be identified in the case of southern or northern hybridization.
18.1 HYBRIDIZATION OF SOUTHERN FILTERS

18.1.1 Prehybridization
1. Prepare prehybridization buffer up to 25 mL with sterile double distilled water and add
to southern filter in a hybridization box or bag.
2. Denature 0.5 mL of 1 mg/mL solution of sonicated non-homologous DNA (Salmon
sperm DNA or calf thymus DNA), by heating to 100°C for 5 minutes. Chill on ice and
add to prehybridization buffer.
3. After sealing the bag or closing the box, dehybridize in a shaking water bath or in a
hybridization oven at 65°C for 1 hour.
4. Denature the labelled probe by heating to 100°C for 5 minutes and chilling on ice and
add the probe to the prehybridization buffer.
5. Allow the hybridization by incubating at least for 12 hours at 65°C.
103
6. Following hybridization, wash the filters by incubating them in buffer containing 2X

SSPE, 0.1% (w/v) SDS at room temperature for 10 minutes. Repeat the step one more
time.
7. Replace the solution with 1X SSPE, 0.1% (w/v) SDS incubate at 65°C for 15 minutes.
8. Replace the solution with 0.1X SSPE, 0.1% (w/v) SDS incubate at 65°C for 10 minutes.
9. Repeat if necessary.
10. Remove the filter from the hybridization box or bag and wrap it with saran wrap and
expose the filter to X-ray film and incubate in –70°C for at least 24 hours and develop
the X-ray film through photographic methods such as, developing, stopping and fixing
and analysing the autoradiogram.
Buffers
(i) 20X SSPE (pH 7.7)
3.6 M NaCl
0.2 M sodium phosphate
0.02 M EDTA
(ii) 100X Denhardt’s solution
2% w/v BSA
2% Ficoll
2% polyvinyl pyrolidone
(iii) Prehybridization buffer
5X SSPE
5X Denhardt’s solution
0.1% (w/v) SDS solution
(iv) Hybridization buffer
5X SSPE
0.1% (w/v) SDS solution
Non-homologous denatured DNA (1 mg/mL)
Labelled denatured probe DNA
(v) Posthybridization wash buffers
Buffer I
2X SSPE
0.1% (w/v) SDS
Buffer II
1X SSPE
0.1% (w/v) SDS
Buffer III
0.1X SSPE
0.1% (w/v) SDS
Hybridization Techniques #
18.2 COLONY HYBRIDIZATION

Protocol
1. Immerse the southern filter in 6X SSC for 2 minutes. Then slip the filter into a heat-
sealable plastic bag and add 15 mL of prehybridization buffer and incubate for 2–4 hours
in a water bath or hybridization oven at 68°C.
2. Then remove the bag from the water bath or hybridization oven and remove the pre-
hybridization solution and add hybridization, containing the denatured labelled DNA
probe and add, seal it and incubate it at 68°C for 12–14 hours.
3. After hybridization remove the filter from the bag and incubate it in a washing solution
containing 2X SSC and 0.5% SDS at room temperature for 5 minutes.
4. After 5 minutes, transfer the filter to a fresh tray containing a solution 2X SSC and 0.1%
SDS and incubate at room temperature for 15 minutes with gentle shaking.
5. Then transfer the filter fresh tray containing a solution 0.1X SSC and 0.5% SDS and
incubate at 68°C for 4–6 hours with gentle shaking.
6. Then dry the filter at room temperature on a Whatman filter paper (3 mm), and expose
the filter to X-ray film and incubate in –70°C for at least 24 hours and develop the
X-ray film through photographic methods such as, developing, stopping and fixing and
analysing the autoradiogram.
Buffers
(i) 20X SSC for 1000 mL
NaCl – 175.3 g
Sodium citrate – 88.2 g
dd.H2O – 800 mL
Adjust pH 7.0 with a few drops of 10/N NaOH and the volume is made up to 1 litre and
autoclaved.
(ii) Denhardt’s solution 50X for 500 mL
Ficoll – 5.0 g
Polyvenyl pyrolidone – 5.0 g
BSA – 5.0 g
H2O up to – 500 mL
Filter through a disposable nalgene filter disperse into 25 mL aliquot and store at –20°C
(iii) Salmon sperm DNA or Calf thymus DNA
100 mg/mL denatured SS DNA or CT DNA
(iv) Prehybridization fluid
6X SSC
100 mg/mL denatured Salmon sperm DNA
(v) Hybridization solution
6X SSC
100 mg/mL denatured Salmon sperm DNA
32
P labelled denatured DNA probe
(vi) Posthybridization wash solution
Solution I 2X SSC and 0.5% SDS
Solution II 2X SSC and 0.1% SDS
Solution III 0.1X SSC and 0.5% SDS
(vi) Development of autoradiogram
(a) Developer for 500 mL
6.6 g from Pack A
44.5 g from Pack B
(b) Stop bath
3% acetic acid or water bath
(c) Fixer for 500 mL
134.0 g in 500 mL
REFERENCES
Born, T.L. and C.G. Miyada (1991), Stained colonies facilitate alignment in non-radioactive
colony hybridization, Bio Techniques, 10 (4), pp. 480–481.
Appendices
APPENDIX
1
Stock Solutions and Working
Concentrations of Antibiotics
Stock solution Working concentration

mg/mL: Storage temp. Minimum Maximum
mg/mL mg/mL
Ampicillin 50 (H2O*) –20°C 20 60–125
Carbenicillin 50 (H2O, 50% EtOH) –20°C 20 60–200
Chloramphenicol 34 (EtOH, MetOH) –20°C 25 170
Gentamycin 10 (H2O) –20°C 15 15
Hygromycin B 10 (H2O) –20°C 10 400
Kanamicin 50 (H2O) +4°C - 10 50
– 20°C
Kasugamycin 10 (H2O) –20°C 1000 1000
Nalidixic acid 5 (H2O, pH to 11 –20°C 15 15
/NaOH)
Neomycin 10 (H2O) –20°C 100 800
Rifampicin** 34 (MetOH) –20°C 150 150
Spectinomycin 10 (H2O) –20°C 100 100
Streptomicin 50 (H2O) –20°C 10 50
Tetracycline*** 5 – 12.5 –20°C 10 50
(EtOH, 70% EtOH) 100
* – Unstable solution, prepare just before use

* * – Light sensitive
*** – Light sensitive, Mg2 Mg2+ – inhibitor
109
110 Appendices
APPENDIX
2
Conversion of rpm to g
The most commonly used standard centrifuge rotor in super speed refrigerated centrifuges such
as Du point, Sorvall and Hitachi is SS-34 type, whose radius is 10.8 cm (4.25 inches). For any
given rpm of the rotor, the g value can be calculated by the following equation:
g = 11.17 ¥ r (rpm/1000)2
when r is the radial distance between the centre of the rotor and the relevant point of the
centrifuge tube and is given in cm. (rmax corresponds to bottom of the centrifuge tube is fixed
angle and swinging bucket rotors and the outermost of the tube in vertical rotors).
For example, if the sample is spun at 10,000 rpm in the above rotors, the g will be
g = 11.17 ¥ 10.8 (10,000/1000)
g = 11.17 ¥ 10.8 (10)2
g = 11.17 ¥ 10.8 ¥ 100 = 12.063
For other rotors and with different radius, the g value can be calculated by using the above
equation.
110
Appendices 111
APPENDIX
3
Conversion of C to rpm
To find out the rpm for a given g value, the following equation is used,
rpm = 1000 ¥ RCF /1117
. ¥r
For example, to find out the correct rpm to get 12,000 g, the calculation is as follows:
rpm = 1000 12,000 rpm /1117
. ¥ 10.8
= 1000 99.5
= 1000 ¥ 9.973 = 9973 rpm
So the samples have to be spun at ~10,000 rpm to get 12,000 g.
111
Appendices
APPENDIX
4
Stock Solutions
1. 10 M Ammonium acetate
Dissolve 385.4 g ammonium acetate in 150 mL H2O
Add H2O to 500 mL
2. Ammonium sulphate, saturated
76 g ammonium sulphate
100 mL H2O
Heat with stirring to just below boiling point
Let stand overnight at room temperature
3. 100 mM ATP
1 g ATP (adenosine triphosphate)
12 mL H2O
Adjust pH to 7.0 with 4 M NaOH
Adjust volume to 16.7 mL with H2O
Store in aliquots indefinitely at –20°C
4. 1 M CaCl2
147 g CaCl2 ◊ 2H2O
H2O to 1 litre
5. Carbonate buffer
1.6 g Na2CO3 (15 mM final)
2.9 g NaHCO3 (35 mM final)
0.2 g NaNO3 (3.1 mM final)
H2O to 1 litre
Adjust to pH 9.5
CAUTION: Sodium azide is poisonous; follow appropriate precautions for handling,
storage, and disposal.
6. DTT (dithiothreitol), 1 M
Dissolve 1.55 g DTT in 10 mL water and filter sterilize. Store in aliquots at –20°C.
112
Appendices !
7. EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0)

Dissolve 186.1 g disodium EDTA dihydrate in 700 mL water. Adjust pH to 8.0 with
10 M NaOH (~50 mL; add slowly). Add water to 1 litre and filter sterilize.
Begin titrating before the sample is completely dissolved. EDTA, even the disodium salt, is
difficult to dissolve at this concentration unless the pH is increased to between 7 and 8.
8. Ethidium bromide 10 mg/mL
Dissolve 100 mg of ethidium bromide in 10 mL dH2O. Wrap tube in aluminium foil and
store at 4°C.
CAUTION: EtBr is extremely mutagenic.
9. 1 M HCl
Mix in the following order:
913.8 mL H2O
86.2 mL concentrated HCl
10. IPTG
IPTG is Isopropyl-b-D-thiogalactopyranoside. Make a 20% (w/v, 0.8 M) solution of
IPTG by dissolving 2 g of IPTG in 8 mL of distilled H2O. Adjust the volume of the
solution to 10 mL with H2O and sterilize by passing it through a 0.22 mm disposable
filter. Dispense the solution into 1 mL aliquots and store them at –20°C.
11. 1 M KCl
74.6 g KCl
H2O to 1 litre
12. LB Media
10 g tryptone
5 g Yeast extract
10 g NaCl
1000 mL water
Adjust pH to 7.5 with 1 M NaOH.
13. LB + Amp
Autoclave and let it cool to less than 60°C. Add 6 mL of ampicillin per litre of sterile LB
media. Do not autoclave the solution containing antibiotics.
14. 1 M MgCl2
20.3 g MgCl2 ◊ 6H2O
H2O to 100 mL
15. 1 M MgSO4
24.6 g MgSO4 ◊ 7H2O
H2O to 100 mL
16. 5 M NaCl
292 g NaCl
H2O to 1 litre
17. NaCl (saline), 0.9% (w/v)
9 g NaCl (154 mM final)
H2O to 1 litre
" Appendices
18. 10 M NaOH
Dissolve 400 g NaOH in 450 mL H2O
H2O to 1 litre
19. PBS (Phosphate-buffered saline)
8.00 g NaCl (0.137 M)
0.20 g KCl (2.7 mM)
0.24 g KH2PO4 (1.4 mM)
1.44 g Na2HPO4 (0.01 M)
H2O to 1 litre
20. 0.1 M Potassium acetate buffer
Solution A: 11.55 mL glacial acetic acid per litre (0.2 M) in water.
Solution B: 19.6 g potassium acetate (KC2H3O2) per litre (0.2 M) in water.
Sterilize the filter if necessary. Store up to 3 months at room temperature.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium
acetate in the same volume. Acetate buffers show concentration-dependent pH changes,
so check the pH by diluting an aliquot of concentrate to the final concentration.
21. Proteinase K - 10 mg/mL in TE
Weigh out 10 mg proteinase K and dissolve in 1 mL TE. The solution can be used
immediately or aliquoted and stored at –20°C.
22. RNase A and RNase T1
Dissolve 100 mg RNase A, if desired. Add 5,000 units of RNase T1 together, in 10 mL
of 10 mM Tris 15 mM NaCl. Boil for 15 minutes and allow to cool slowly to room
temperature. Distribute 1 mL aliquote into 1.5 mL MFT, and store at –20°C.
23. SDS, 20% (w/v)
Dissolve 20 g SDS (sodium dodecyl sulphate or sodium lauryl sulphate) in H2O to
100 mL total volume with stirring. Filter sterilizes using a 0.45-mm filter.
24. SDS electrophoresis buffer, 5X
15.1 g Tris base
72.0 g glycine
5.0 g SDS
Distilled, deionized H2O to 1 litre
Store up to 1 month at 0° to 4°C
Dilute to 1X before use
Do not adjust the pH of the stock solution; the pH is 8.3 when diluted to 1X.
Use purified SDS if appropriate.
25. SDS sample buffer
Ingredient 2X 4X Final conc.

in 1X buffer
0.5 M Tris-Cl, pH 6.8a 2.5 mL 5.0 mL 62.5 mM
SDS 0.4 g 0.8 g 2% (w/v)
Glycerol 2.0 mL 4.0 mL 10% (v/v)
Appendices #
Bromphenol blue 20 mg 40 mg 0.1% (w/v)
2-Mercaptoethanol b,c 400 mL 800 mL ~300 mM
H2 O to 10 mL to 10 mL –
a
See recipe below.
b
Alternatively, dithiothreitol (DTT), at a final concentration of 100 mM, can be substituted
for 2-mercaptoethanol.
c
Add just before use.
26. Sodium acetate, 3 M
Dissolve 408 g sodium acetate trihydrate (NaC2H3O2 ◊3H2O) in 800 mL H2O.
Adjust pH to 4.8, 5.0, or 5.2 (as desired) with 3 M acetic acid. Add H2O to 1 litre.
Sterilize the filter.
27. 0.1 M Sodium acetate buffer
Refer to the following table (Preparation of 0.1 M Sodium acetate buffer at different pH).
For desired pH, mix the indicated volumes of solutions A and B, then dilute with water
to 100 mL. Filter sterilize, if necessary. Store up to 3 months at room temperature.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium
acetate in the same volume. Acetate buffers show concentration-dependent pH changes,
so check the pH by diluting an aliquot of concentrate to the final concentration.
Preparation of 0.1 M Sodium acetate buffer at different pH
pH Solution A Solution B
(vol. of 0.1 M acetic acid ) (vol. of 0.1 M sodium acetate)
3 982.3 mL 17.7 mL
4 847.0 mL 153.0 mL
5 357.0 mL 643.0 mL
6 52.2 mL 947.8 mL
28. 0.1 M Sodium phosphate buffer

Solution A: 27.6 g NaH2PO4◊H2O per litre (0.2 M final) in water.
Solution B: 53.65 g Na2HPO4◊7H2O per litre (0.2 M) in water.
Referring to the Table 10.2 for desired pH, mix the indicated volumes of solutions A and
B, then dilute with water to 200 mL. Sterilize the filter, if necessary. Store up to 3 months
at room temperature. This buffer may be made as a 5- or 10-fold concentrate by scaling
up the amount of sodium phosphate in the same final volume. Phosphate buffers show
concentration-dependent changes in pH, so check the pH by diluting an aliquot of the
concentrate to the final concentration.
29. TBS (Tris-buffered saline)
100 mM Tris-Cl, pH 7.5 (see recipe below)
0.9% (w/v) NaCl
Store up to several months at 4°C
30. 1 M Tris-Cl
Dissolve 121 g Tris base in 800 mL H2O
$ Appendices
Adjust to desired pH with concentrated HCl

Adjust volume to 1 litre with H2O
Sterilize the filter if necessary
Store up to 6 months at 4°C or room temperature
Approximately 70 mL HCl is needed to achieve a pH 7.4 solution, and ~42 mL for a
solution that is pH 8.0.
31. X-gal
5-bromo-4-chloro-3-indolyl-b-D-galactoside (same recipe for X-phosphate)
Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a
concentration of 20 mg/mL solution. Use a glass or polypropylene tube. Wrap the tube
containing the solution in aluminum foil to prevent damage by light and store at –20°C.
It is not necessary to sterilize X-gal solutions.
Appendices 117
APPENDIX
5
DNA/Protein Conversions
1 kb of DNA = 333 amino acids approx. is 3.7 ¥ 10E4 Da

10 kDa protein approx. is 270 bp DNA
30 kDa protein approx. is 810 bp DNA
50 kDa protein approx. is 1.32 kb DNA
100 kDa protein approx. is 2.7 kb DNA
117
118 Appendices
APPENDIX
6
Common Conversions of Oligonucleotides
Molecular weight
MW = 333 ¥ N
Concentration of oligonucleotides
C (mM or pmol/mL) = A260/(0.01 ¥ N)
C (ng/mL) = (A260 ¥ MW)/(0.01 ¥ N)
MW = molecular weight, Da
A260 = absorbance at 260 nm
N = number of bases
Melting temperature of Duplex DNA and oligonucleotides
For Duplex oligonucleotide shorter than 25 bp, “The Wallace Rule”
Tm (in °C) = 2(A + T) + 4(C + G)
where,
(A + T) – the sum of the A and T residues in the oligonucleotide,
(C + G) – the sum of G and C residues in the oligonucleotide.
Presence of m 5C in oligonucleotide increases melting temperature of duplex.
Presence of m 4C or m 6A decreases melting temperature.
For Duplex DNA, < 100 bp long
Tm (in °C) = 81.5°C + 16.6(log10 [Na+]) + 0.41(% [G + C]) – 675/n-1.0 m,
where
n = Number of bases in the oligonucleotide
m = the percentage of base-pair mismatches
118
Appendices 119
COMMON CONVERSIONS OF NUCLEIC ACIDS

Molar conversions
1 mg of 1000 bp DNA = 1.52 pmol
1 mg of pUC18/19 DNA (2686 bp) = 0.57 pmol
1 mg of pBR322 DNA (4361 bp) = 0.35 pmol
1 mg of SV40 DNA (5243 bp) = 0.29 pmol
1 mg of PhiX174 DNA (5386 bp) = 0.28 pmol
1 mg of M13mp18/19 DNA (7250 bp) = 0.21 pmol
1 mg of lambda phage DNA (48502 bp) = 0.03 pmol
1 pmol of 1000 bp DNA = 0.66 mg
1 pmol of pUC18/19 DNA (2686 bp) = 1.77 mg
1 pmol of pBR322 DNA (4361 bp) = 2.88 mg
1 pmol of SV40 DNA (5243 bp) = 3.46 mg
1 pmol of PhiX174 DNA (5386 bp) = 3.54 mg
1 pmol of M13mp18/19 DNA (7250 bp) = 4.78 mg
1 pmol of lambda phage DNA (48502 bp) = 32.01 mg
Spectrophotometric conversions
1 A260 of dsDNA = 50 mg/mL = 0.15 mM (in nucleotides)
1 A260 of ssDNA = 33 mg/mL = 0.1 mM (in nucleotides)
1 A260 of ssRNA = 40 mg/mL = 0.12 mM (in nucleotides)
1 mM (in nucleotides) of dsDNA = 6.7 A260 units
1 mM (in nucleotides) of ssDNA = 10.0 A260 units
1 mM (in nucleotides) of ssRNA = 8.3 A260 units
The average MW of a deoxyribonucleotide base = 333 Daltons
The average MW of a ribonucleotide base = 340 Daltons
120 Appendices
APPENDIX
7
Restriction Enzymes and their
Cleavage Sites
Restriction enzyme Recognition site Restriction enzyme Recognition site

Aat II GACGIGC BssH II GG CGCGC
AccI GTG (A/T)(T/G)AC Bst71 I GCAGC(8/12)
AccIll TGCCGGA Bst98 I CG TTAAG
Acc65 I GG GTACC Bst E II GG GTNACC
AccB7 I CCANNNNG NTGG Bst O I CCG (A/T)GG
AcyI G(A/G)G CG(T/C)C Bst X I CCANNNNNG NTGG
Age l AG CCGGT Bst Z I CG GGCCG
Alu l AGG CT Bsu36 I CCG TNAGG
A/w26 I GG TCTC(1/5) Cfo I GCGG C
A/w441 GG TGCAC Cla l ATG CGAT
Apa l GGGCCG C Csp I CGG G(A/T)CCG
Ava I CG (T/C)CG(A/G)G Csp 45 I TTG CGAA
Ava lI GG G(A/T)CC Dde I CG TNAG
Ba/I TGGG CCA Dpn I GmeAG TC
BamH l GG GATCC Dra l TTTG AAA
Ban I GG G (T/C)(A/G)CC EclHK I GACNNNG NNGTC
Ban II G(A/G)GC(T/C)G C Eco47 III ACGG GCT
Bbu l GCATGG C Eco52 I CG GGCCG
Bc/I TG GATCA Eco72 I CACG GTG
Bgl l GCCNNNNG NGGC EcoI CR I GAGG CJC
Bg/Il AG GATCT EcoR I GG AATTC
BsaM I GATTGCNG EcoR V GATG ATC
BsaO I CG(A/G)(T/C)G CG Fok I GGATG(9/13)
Bsp1286 I G(G/A/T)GC(C/A/T)G C Hae ll (A/G)GCGCG (T/C)
BsrBR I GATNNG NNATC HaelIl GGG CC
BsrS I ACTGGNG Hha I GCGG C
(Contd.)
120
Appendices 121
(Contd.)
Restriction enzyme Recognition site Restriction enzyme Recognition site

Hinc II GT(T/C)G (A/G)AC Rsa l GTG AC
Hind III AG AGCTT Sac I GAGGCTG C
Hinf I GG ANTC Sac II CCGCG GG
Hpa I GTTG AAC Sal l GG TCGAC
Hpa II CG CGG Sau3A I GGATC
Hsp92 I G(A/G)G CG(T/C)C Sau96 I GG GNCC
Hsp92 II CATGG Sca l AGTG ACT
I-Ppo I CTCTCTTAAG GGTAGC Sfi I GGCCNNNNGNGGCC
Kpn I GGTACG C Sgf I GCGATG CGC
Mbo I GGATC Sin I GG G(A/T)CC
Mbo II GAAGA(8/7) Sma l CCCG GGG
Mlu l AG CGCGT SnaB I TACG GTA
Msp I CG CGG
Spe l AG CTAGT
MspA I C(A/C)GG C(G/T)G
Sph I GCATGG C
Nae l GCCG GGC
Ssp l AATG ATT
Nar GGG CGCC
Stu l AGGG CCT
Nci I CCG (G/C)GG
Sty l CG C(A/T)(T/A)GG
Nco I CG CATGG
Taq I TG CGA
Nde l CAG TATG
Tru9 I TG TAA
NgoM I GG CCGGC
Tthlll I GACNG NNGTC
Nhe I GG CTAGC
Not I GCG GGCCGC Vsp I AG TAAT
Nru I TCGG CGA Xba I TG CTAGA
Nsi l ATGCAG T Xho I CG TCGAG
Pst l CTGCAG G Xho II (A/G)G GATC(T/C)
Pvu l CGATG CG Xma l CG CCGGG
Rvu II CAGG CTG Xmn I GAANNG NNTTC
Appendices
APPENDIX
8
Estimation of Ends (3¢¢ or 5¢¢)
Concentration
Circular DNA
pmol ends = pmol DNA ¥ number of cuts ¥ 2
Linear DNA
pmol ends = pmol DNA ¥ (number of cuts ¥ 2 + 2)
1 mg of 1000 bp DNA = 3.04 pmol ends
1 mg of linear pUC18/19 DNA = 1.14 pmol ends
1 mg of linear pBR322 DNA = 0.7 pmol ends
1 mg of linear SV40 DNA = 0.58 pmol ends
1 mg of linear PhiX174 DNA = 0.56 pmol ends
1 mg of linear M13mp18/19 DNA = 0.42 pmol ends
1 mg of lambda phage DNA = 0.06 pmol ends
DNA size migration with sample loading dyes

Agarose Xylene cyanol FF Bromophenol blue Orange G
concentration, %
0.7–1.7 ~4000 bp ~300 bp ~50 bp
2.5–3.0 ~800 bp ~100 bp ~30 bp
122
Appendices 123
APPENDIX
9
Recommended Gel Percentages for
Separation of Linear DNA
Agarose Range of separation Polyacrylamide gel (%) Range of separation (bp)

gel (%) (bp)
0.5 1,000–30,000 3.5 100–1,000
0.7 800–12,000 5.0 80–500
1.0 500–10,000 8.0 60–400
1.2 400–7,000 12.0 40–200
1.4 200–4,000 20.0 5–100
123
124 Appendices
APPENDIX
10
Calculating Primer Quantity
Conversion to absolute quantity (in pmole)

weight in mg ¥ 1,000,000
Primer in pmole =
Length ¥ 327
Example: 0.1 mg of 20 oligomer
0.1 ¥ 1,000,000
= 15.3 pmole primer
20 ¥ 327
Conversion to weight (in m g)
Pmol ¥ length ¥ 327
Weight in mg =
1,000,000
Example: 10 pmol of 25 oligomer
10 ¥ 25 ¥ 327
= 0.081 mg primer
1,000,000
124
Laboratory Manual for
Genetic Engineering
S. John Vennison
This systematically designed laboratory manual elucidates a number of techniques which help the students
carry out various experiments in the field of genetic engineering.
The book explains the methods for the isolation of DNA and RNA as well as electrophoresis techniques for
DNA, RNA and proteins. It discusses DNA manipulation by restriction digestion and construction of
recombinant DNA by ligation. Besides, the book focuses on various methodologies for DNA transformation
and molecular hybridization. While discussing all these techniques, the book puts emphasis on important
techniques such as DNA isolation from Gram positive bacteria including Bacillus sp., the slot-lysis electro-
phoresis technique which is useful in DNA profile analysis of both Gram negative and positive bacteria,
plasmid transduction in Bacillus sp., and the conjugal transfer of plasmid DNA in cyanobacteria, Bacillus and
Agrobacterium tumefaciens.
This book is intended for the undergraduate and postgraduate students of biotechnology for their laboratory
courses in genetic engineering. Besides, it will be useful for the students specializing in genetic engineering,
molecular biology and molecular microbiology.
KEY FEATURES
l Includes about 60 different experiments.
l Contains several figures to reinforce the understanding of the techniques discussed.
l Gives useful information about preparation of stock solutions, DNA/protein conversions, restriction
enzymes and their recognition sequences, and so on in Appendices.
THE AUTHOR
S. John Vennison, Ph.D., is Lecturer in the Department of Biotechnology, Anna University, Tiruchirappalli. He
has more than 10 years of teaching experience and 18 years of research experience in the field of genetic
engineering. He has published several research papers in the national and international journals. His research
areas include molecular biology and biotechnology. A recipient of the Visiting Scientist Award, 1995 by
UNESCO, Dr. Vennison is a life member of Biotech Research Society of India.
ISBN:978-81-203-3814-2
www.phindia.com 9 788120 338142

Laboratory Manual For Genetic Engineering

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Laboratory Manual For Genetic Engineering

Uploaded by

Copyright:

Available Formats

Laboratory Manual

Preface .......................................................................................................................................................................... vii

2.13 Ultra Purification of Plasmid DNA through Cesium Chloride

9. Purification of DNA from Agarose and Polyacrylamide Gels ............................... 55-56

16.3 Southern Blotting 94

1.1 TOTAL DNA EXTRACTION FROM AGROBACTERIUM

1.2 TOTAL CELL DNA ISOLATION FROM

(v) DNA dissolving and storage buffer

1.3 GENOMIC DNA ISOLATION FROM BLOOD

1.4 A RAPID PROCEDURE FOR ISOLATION OF

2.1 GROWTH OF BACTERIA

TABLE 2.1 Plasmid growth and replication

TABLE 2.1 Plasmid growth and replication (Contd.)

2.2 HARVESTING AND LYSING OF BACTERIA

2.3 PURIFICATION OF THE PLASMID DNA

2.3.1 Cloning in Plasmid Vectors

2.3.2 Cloning DNA Fragments with Protruding Ends

2.4 MODIFIED ALKALINE LYSIS PROTOCOL FOR BOTH

2.5 HIGH MOLECULAR WEIGHT PLASMID PREPARATION

2.6 PLASMID ISOLATION FROM E. COLI (MINI PREPARATION)

7. Add 100 mg of cesium chloride (CsCl)

2.7 LARGE SCALE PLASMID PREPARATION FOR

2.7.1 Plasmid Amplification

2.7.3 Lysis with Alkali

2.8 RAPID BOILING METHOD OF ISOLATION OF

2.9 ISOLATION OF PLASMID DNA FROM

2.10 PURIFICATION OF HIGH MOLECULAR WEIGHT PLASMIDS

2.11 ISOLATION OF PLASMID DNA FROM BACILLUS SUBTILIS AND

2.12 PURIFICATION OF GENOMIC AND PLASMID DNA THROUGH

3. Transfer the upper aqueous phase to another sterile Eppendorf.

2.13 ULTRA PURIFICATION OF PLASMID DNA THROUGH CESIUM

Figure 2.3(b) Recovery of plasmid DNA band by inserting a hypodermic syringe.

Recovered plasmid DNA in CsCl

Figure 2.3(c) Recovered circular plasmid DNA in a hypodermic syringe.

3.1 ISOLATION OF BACTERIAL RNA

Figure 3.1 Agarose gel electrophoresis of RNA.

3.2 ISOLATION OF mRNA FROM CULTURED MAMMALIAN CELLS

20. Centrifuge for 10 minutes in an Eppendorf centrifuge tube.

4.1 ESTIMATION OF DNA

3. Set wavelength at 260 nm

4.1.2 TD-20/20 Luminometer Method for DNA Quantitation

∑ 2 ¥ 500 u, T4 DNA polymerase (5–10 u/mL)

TABLE 4.1 Volume of master mix components required by number of reactions

made using 10 mM Tris-HCl (pH 7.3–7.5) or TE buffer (10 mM Tris, 1 mM EDTA

4.1.3 Diphenylamine Method

DNA stock solution

4.2 ESTIMATION OF RNA

Restriction enzyme digestion is performed by incubating double-stranded DNA molecules with

5.1 RESTRICTION DIGESTION OF DNA

5.2 PURIFICATION OF RESTRICTED DNA FRAGMENTS

5.3 DNA LIGATION

3. Transform the ligated DNA in to E. coli either by competent cell transformation or by

6.1 IMPORTANT PARAMETERS IN THE PCR

6.1.3 Length of Expected Product

6.2 POLYMERASE CHAIN REACTION (PCR)

Figure 6.1 Ethidium bromide-stained PCR products after gel electrophoresis.

6.3 RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)

Figure 6.2 Ethidium bromide-stained RAPD products after gel electrophoresis.

7.1 AGAROSE GEL ELECTROPHORESIS OF DNA

7.1.1 The Rate of Migration of DNA through Agarose Gels

The molecular size of the DNA

The concentration of agarose

The conformation of the DNA