Handbook of Practical Protocols Second Edition

HANDBOOK OF PRACTICAL PROTOCOLS
Second Edition

Second Edition
Amit Yadav.
Basic techniques in Genetic Engineering

DNA Fingerprinting (basics of markers, ribotyping and genotyping);
Cloning (Ligation and Transformation);
PCR (Basics, types of, primer designing and applications),
Plasmid Extraction and
Agarose Gel Electrophoresis
Contents
1. Before you start!
2. Stocks and Media
3. Restriction Fragment length Polymorphism (RFLP)
4. Agarose Gel Electrophoresis
5. Ligation, Competent cell preparation, Transformation and Plating
6. Polymerase Chain Reaction (PCR) and Primer Designing
7. Plasmid Isolation from E. coli
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Before You Start!
IMPORTANT: Make sure that you have gathered all required chemicals, lab-ware and
Equipments before staring any experiment. Please read the following check list.
A. RFLP
1. Restriction enzyme, 1U/ul (e.g. EcoRI, Hind III or as per experiment design)
2. Restriction enzyme buffer (2X)
3. Nuclease free water
4. Target DNA (1ug/ul)
5. Incubation chamber at desired temperature (mostly at 370C)
6. Equipments and chemicals related to Agarose Gel Electrophoresis (see below)
B. PCR
1. Taq Polymerase (1U/ul) 5. Forward primer (10mM)
2. Taq Polymerase Buffer (10X) 6. Reverse primer (10mM)
3. MgCl2 or KCL (25mM) 7. Nuclease free water
4. dNTP mix (20mM) 8. PCR machine
9. Equipments and chemicals related to Agarose Gel Electrophoresis (see below)
C. Ligation, Competent cell preparation, Transformation and Plating

1. Ligase enzyme (1U/ul) 4. Agar powder
2. Ligase buffer (2X) 5. Ampicilin (10mg)
3. Calcium Chloride, Pure
6. Luria-Bertani (LB) media (1%Trytone, 0.5%Yeast Extract, 1%NaCl) at pH 7.2 to 7.4
D. Plasmid Isolation
1. Sodium dodecyl or lyuryl Sulphate (SDS)
2. EDTA (Ethylene Di-amine Tetra Acetic acid)
3. Tris-HCl or Tris Base 7. Potassium acetate (5M)
4. Glucose 8. Sodium acetate (3M)
5. Lysozyme (10mg/ml) 9. Iso-propanol
6. NaOH 10. Absolute alcohol
11. RNase enzyme (ready to use)
E. Agarose Gel Electrophoresis

1. Agarose powder 3. DNA marker
2. Ethidium bromide (10mg/ml) 4. TAE Buffer at pH 8.0
5. Casting trays, Combs, Electrophoresis Tank, Power Pack and electricity!! 
6. UV Trans-illuminator
F. General Plastic ware and Equipments

1. Refrigerated centrifuge,
2. Incubator Shaker (370C),
3. Petri plates, Conical flask, Spreader, Measuring cylinders, Culture tubes, Test tube rack;
4. MicroPipettes, tips,
5. Distilled water, Crushed ice,
6. Water bath, Dry bath
7. Eppendorffs 1.5ml, 2ml; PCR Tubes, Eppendorff racks; PCR tube racks
8. -200C Mini Chillier to keep enzymes,
9. Laminar Air flow,
10. Burner/ Spirit lamp, Match Box, other general lab-ware
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Stocks and Media
1. LB Broth: 1% Tryptone, 0.5% Yeast Extract, 1% NaCl at pH 7.2 to 7.4 OR Dissolve 1.5
g of LB broth powder (readymade) in 40 ml of distilled water. Make up the volume to 60
ml. Set pH at 7.2 to 7.4. Autoclave before use. Prepare 50 ml of LB broth in a 250 ml
conical flask for growing the cells. Remaining 10 ml LB broth is used as 5 ml in 30 ml
capacity test tube.
2. Preparation of plates with Ampicillin: Melt the LB agar (autoclaved) and cool till the
temperature is about 40degC- 50degC. Remember, ampicillin degrades at 55degC. Pour
15 to 20ml in one plate and for the remaining LB agar add Ampicillin (10mg/ul- stock;
100ug/ul final conc.), Mix well and pour media into plates.
3. TAE Buffer (pH 8.0): 40mM Tris, 1mM EDTA. Adjust the pH with acetic acid.
Autoclave.
4. Ethidium Bromide (10mg/ml): Dissolve EtBr in ddwater, stir overnight, store in dark
coloured bottle. CAUTION: Suspected carcinogenic! Store at RT.
5. Ampicillin (10mg/ml): Dissolve ampicillin in very little volume of dilute NaOH, add
ddwater to make the volume. Store at -20degC. CAUTION: Suspected carcinogenic!
6. Lysozyme (10mg/ml): Dissolve Lysozyme in 10mM Tris-HCL (pH 8.0). Store at RT.
7. TEG Buffer (pH 8.0): 25mM Tris, 10mM EDTA & 50mM Glucose. Adjust pH with
HCL. Autoclave.
8. 5M Potassium acetate (49.07gm in 100ml water). Autoclave.
9. 3M Sodium Acetate (40.83gm in 100ml water). Autoclave.
10. TE Buffer (pH 8.0): 10mM Tris, 1mM EDTA. Adjust the pH with HCL. Autoclave.
11. NaOH- SDS Solution: 2N NaOH and 10% SDS. Take 5ml each and dilute to 50ml.
Prepared just before use. Do not autoclave.
12. 20mM Calcium chloride (1.47gm in 100ml). Keep in crushed ice before use. Do not
autoclave. Filter sterilise.
Standard Metric System of measurements: Know it well, don’t get confuse!
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Restriction Fragment Length Polymorphism (RFLP)
Introduction:
Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may
be differentiated by analysis of patterns derived from cleavage of their DNA. If two
organisms differ in the distance between sites of cleavage of a particular restriction
endonuclease, the length of the fragments produced will differ when the DNA is digested with
a restriction enzyme. The similarity of the patterns generated can be used to differentiate
species (and even strains) from one another. Restriction fragment length polymorphisms
(RFLPs) were the first type of molecular markers used in linkage studies. RFLPs arise
because mutations can create or destroy the sites recognized by specific restriction enzymes,
leading to variations between individuals in the length of restriction fragments produced from
identical regions of the genome.
1. They are co-dominant and unaffected by the environment;

2. Any source DNA can be used for the analysis;
3. Many markers can be mapped in a population that is not
stressed by the effects of phenotypic mutations.
Diagrammatic representation 
Ribotyping (RFLP of rRNA genes) is a sensitive, highly discriminatory subtyping technique

that is effective for prokaryotes. It generates DNA fingerprints useful in identifying,
monitoring and tracking for selected microbial strains within an environment. Detection is
based on analysis for rRNA gene fragments generated by subjecting genomic DNA to
digestion by restriction endonucleases.
Ribotyping is able to differentiate between different strains within a species or subspecies,

because the locations of various restriction enzyme recognition sites within specific genetic
loci are often polymorphic from strain to strain. For different strains, the technique results in
the endonucleases cutting genomic DNA to different fragment lengths for individual genes,
generating distinct banding patterns, which is termed 'restriction fragment length
polymorphisms' (RFLP). Even though a myriad of DNA fragments are generated due to all of
the genes present, RFLP analysis for ribotyping is appropriately detailed and specific for
analysis, because the oligonucleotide probes target only rRNA genes, which are highly
conserved in nature. The fingerprint patterns generated for individual strains by ribotyping are
stored in libraries. Sample analysis consists of careful, critical comparison of the patterns. To
speed analysis, some knowledge of the genus, species or source of organism in question is
helpful to select the patterns to be considered for comparison.
Genotyping refers to the process of determining the genotype of an individual by the use of
biological assays. Current methods of doing this include RFLP, AFLP, PCR, DNA
sequencing and hybridization to DNA microarrays or beads. The technology is important in
clinical research for the investigation of disease-associated genes.
Due to current technological limitations, almost all genotyping is partial. That is, only a small
fraction of an individual’s genotype is determined. Phenotype may also be influenced by
epigenetic factors such as DNA methylation. Genotyping applies to a broad range of
"individuals" including microorganisms. Viruses for instance, or bacteria, can be genotyped.
Genotyping in this context may help in controlling the spreading of pathogens, by tracing the
origin of outbreaks. This area is often referred to as molecular epidemiology or forensic
microbiology.
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The "individuals" can also be human beings. When testing for father-/motherhood for
instance, scientists typically only need to look at 10 or 20 genomic regions (like Single
nucleotide polymorphism (SNPs) to determine relationship or lack thereof. That is a tiny
fraction of the human genome, which consists of three billion or so nucleotides.
Applications:
1. RFLP analysis of a family can detect the segregation of an RFLP that can be used to test
for statistically significant linkage to the allele for an inherited disease or some other
human trait of interest screening human DNA for the presence of potentially deleterious
genes.
2. Providing evidence to establish the innocence of, or a probability of the guilt of, a crime
suspect by DNA fingerprinting RFLP is suited for construction of linkage maps.
Precautions to be taken before stating experiment:

1. Switch on the dry or water bath, before starting and set the temperature to 37degC.
2. The staining dye contains Ethidium Bromide. This is known to be carcinogenic and hence
should be handled with extreme care. Use gloves while handling the staining dye and the
Agarose gel.
3. Enzymes are sensitive to temperature. Always keep the enzymes at -20degC.
4. Prepare 1% agarose gel before setting up the restriction digestion reaction. All the
agarose gel to set for 1hr at room temperature.
Procedure:
PART A: Restriction Digestion, Setting up of Restriction digestion reaction:
1. Three reference samples and one unknown sample is digested with HindIII restriction
enzyme.
2. Four separate reactions have to be set up.
3. Remove the Reference samples, unknown DNA samples; assay buffer kept in -200 C and
put them on crushed ice. Allow the components to get thawed on ice.
4. Prepare the reaction mix as given below. Add all the components in the same order as
given below:
DNA sample 15.0 μl (~1ug to 5ug)
10 X Assay buffer 3.0 μl
Hind III enzyme (5U/μl) 1.5 μl
Water 10.5 μl
Total 30.0 μl
5. Add all the components for the mix and tap it 2-3 times for mixing. Give a short spin at
10000 rpm for 20 sec
6. Incubate the mix at 37degC dry bath for 1 hour.

Run 1% agarose gel as described later to visualise the isolated plasmids.
Observations:
Observe the DNA banding pattern obtained by three different reference samples
and one unknown DNA sample. Compare the unknown sample with that of the
reference sample.
Interpretation: 1. Reference Sample 1

1. From the restriction profiles obtained, one can note that restriction 2. Reference Sample 2
profile of unknown sample matches with the reference sample 2. 3. Reference Sample 3
4. Unknown Sample
2. The restriction enzyme recognizes and cuts only a particular base 5. 1kb DNA ladder
sequence unique to it.
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3. Even a slight change occurring in the nucleotide bases will results in loss of recognition
site for a particular restriction enzyme. Hence giving rise to different banding pattern.
4. RFLP technique helps in identifying a particular trait of an organism that can be linked to
a few mutations in the restriction enzyme target sequence.
Polymerase Chain Reaction (PCR)
Introduction:
1. The Polymerase Chain Reaction (PCR) invented by Kary Mullis in 1983, is a primer-
mediated enzymatic amplification of specifically cloned or genomic DNA sequences.
2. This PCR process, invented more than a decade ago, has been automated for routine use
in laboratories worldwide. The template DNA contains the target sequence, which may be
tens or thousands of nucleotides in length.
3. A thermostable polymerase such as Taq DNA Polymerase (purified from Thermus
aquaticus) catalyzes the buffered reaction in which an excess of oligo-nucleotide primer
pair and four deoxynucleoside triphosphates (dNTPs) are used to make millions of copies
of the target sequence.
4. PCR technique has been modified to suit various applications. Some types of PCR
includes: nested PCR, multiplex PCR, inverse PCR, RT-PCR, Assembly PCR,
Asymmetric PCR, Real time PCR, touch down PCR, Hot-start PCR, colony PCR, etc.
5. The Tm of a primer is defined as the temperature at which half of the primer binding sites
at the DNA template are occupied.
6. PCR mainly involves three basic steps, DNA denaturation, primer annealing and primer
extension. As a heat-resistant Polymerase is used for extension reaction, the reaction can
be repeated continuously without addition of more enzymes. Each cycle doubles the copy
number of the amplified gene: ten cycles produces 2 4 8 16 32 64 128
256 512 1,024= (210) copies. Thus, 30 cycles yields a (210x3) = 109 -fold
amplification.
7. Amplification of template DNA depends on factors like, Mg+ ion concentration in the
buffer, annealing temperature of the primers, repeat sequence in the template, Secondary
structure in template DNA, PCR inhibitors present in template etc.
Procedure:
1. Add the following components to the PCR tube provided in the given order.
Component Stock Working Volume Final
Stock Conc.
Nuclease free water -- -- 17μl --
Taq Polymerase Assay buffer 10X 10X 2.5μl 1X
MgCl2 25mM 25mM 1μl 1mM
dNTPs (of each dNTP)* 100mM 25mM 0.5μl 500μM
Forward Primer# 100μM 10μM 1μl 0.4μM
Reverse Primer# 100μM 10μM 1μl 0.4μM
DNA (Template)++ Custom ~100ng 1μl 100ng
Taq DNA Polymerase 3U/μl 3U/μl 1μl 1-3U/Reac
Total reaction volume 25 μl
*Mix each dNTP (100mM stock) in equal amount to have working stock of 25mM, i.e. concentration of each dNTP
is 25mM; the final concentration of dNTPs can vary from minimum of 200μM to 500μM.
#
Always prepare primer stock of concentration 100μM and working stock of 10μM to achieve 0.4 to 1μM final
concentration.
++
Concentration of DNA used as template depends upon its purity and molarity i.e. size and copy number of DNA
molecule. Prepare working stock of ~100ng from custom conc. of mother stock analysed spectrophotometrically.
2. Mix the components gently; a short spin may be given at 10000 rpm for 20sec.
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3. Layer the reaction mix with mineral oil, if needed (depending on the Thermal cycler /
PCR machine used). Place the PCR tubes in the thermal cycler machine.
Reaction Conditions:
Set the PCR conditions in the machine and start the reaction. Conditions for amplification of 1 kb
DNA fragment is as follows:
Initial denaturation 94°C for 2 min

Denaturation 94°C for 30 sec
Annealing 60°C for 30 sec 35 Cycles
Extension 72°C for 30 sec
Final extension 72°C for 1min
Hold 4°C forever

Run 1% agarose gel as described before to visualise the isolated plasmids.
Interpretation:
Observe the 1 kb fragment amplified on the gel by comparing with the marker. Look for non-
specific bands, if any. Also, look for template DNA and the left over primers. Template DNA
would show up only if it’s added more. No primers or primer-dimers would be left over if
amplification was good. Non-specific bands would show up only if the primer(s) bind other
regions other than the specific region. Increasing annealing temperature often helps in reducing
non-specific amplification.
Why some PCRs fail?

Not enough primers added (calculate the primer contribution in the final product. For 5 μg of 100
bp PCR product, one would need to use 1.25 μg each of 25 base long primers); Not enough dNTP
mix added (calculate the μg of it used and μg of product expected); Template is GC-rich (>65%
GC-rich templates are usually not amplified by Taq Polymerase); Long fragment is amplified
(different enzyme mixes available for PCR of up to 35 kb, Taq pol alone can amplify only till ~3
kb); Primer has mismatches with the template (last base mismatch would generate only ~10%
PCR product); PCR inhibitors present in samples (like blood, fibrous leaves, etc.)
Why thermophilic enzymes required for PCR?

Same enzyme added can survive through multiple cycles of denaturation and thus, no further
additions are required between cycles.
How PCR reaction cycle parameters are decided?

Based on length of the product to be amplified, Tm value of the primer, GC composition of the
template DNA, etc
GENE CLONING: Ligation, Competent cell preparation, Transformation and Plating
Introduction:
1. Cloning is the creation of an exact genetic replica of a small segment of DNA, a cell or a
whole organism.
2. The development of techniques for transferring pieces of DNA from one organism to
another in the early 1970s has led to today’s recombinant DNA technologies, which
directly manipulate genetic material to create organisms with tailor-made genetic make-
ups to perform specific tasks.
3. Gene cloning involves the following basic steps: Isolated pure DNA (usually by PCR) is
cut into small gene portions by restriction endonucleases. In a similar way plasmids are
treated with the same endonucleases leaving reactive cut ends in the plasmid structure.
The excised DNA sections are then mixed with the opened plasmids in the presence of
another joining enzyme—Ligase, which can anneal or join the ends of the DNA fragment
and the plasmid. These combined DNA structures are introduced into host cells (e.g.,
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bacteria or yeasts) by a process called transformation. Once inside the host cells, the
recombinant DNA plasmids replicate many times, thus providing many copies of each
donor DNA fragments.
Applications:
1. Cloning is essential to enable enough copies of the gene to be analyzed to find genes.
This may lead to human genetic testing to diagnose genetic conditions and predictive
or pre-symptomatic genetic testing for genetic conditions in asymptomatic
individuals or prenatally. In addition, forensic genetic testing relies on the ability to
understand the information in the non-coding regions of the DNA, i.e. those areas
that do not contain genes.
2. Gene cloning is also important for the development of drugs and treatments such as
in pharmaco-genetics and gene therapy originally rDNA was grown principally in
simple microorganisms such as bacteria and yeast. However, novel genetic material
can now be introduced into higher animals and plants and this is resulting in new
developments in agricultural sciences in particular.
3. Basically there are three types of cloning technologies i.e., recombinant DNA
technology or DNA cloning, reproductive cloning and therapeutic cloning.
4. DNA Cloning: transfer of a DNA fragment of interest from one organism to a self-
replicating genetic element such as a bacterial plasmid. The DNA of interest can then
be propagated in a foreign host cell
5. Reproductive cloning used to generate an animal that has the same nuclear DNA as
another currently or previously existing animal. The purpose of this type of cloning is
to produce a genetic duplicate of an existing or previously existing organism Dolly
was created by reproductive cloning technology. The technology can be used to
mass-produce animals with special qualities, such as animals that are important
agriculturally or are able to produce helpful drugs for human use.
6. Therapeutic Cloning (biomedical cloning, embryo cloning): mimics the natural
process of producing identical twins or triplets. The purpose of this method of
cloning to enable the correction of health problems.
Precautions:
1. All the procedures should be done under aseptic conditions.
2. All the vials (enzyme & Buffer) are to be spun before use (short spin 10000rpm).
3. Prepare competent cells before starting the cloning experiments
4. Carry out the transformation experiments as soon as the competent cells are prepared.
Do not store the competent cells. (Storage of the CCs can be done only at -70degC for the
period of 3 weeks)
5. Pre-cool all the required reagents (vials, competent cell solution, tips etc)
Procedure:
Day 1: Initiation of E. coli culture and Ligation
1. To the lyophilized host cells add 200μl of LB suspend the cells using pipette. Incubate at
37degC overnight.
2. Setting up of Ligation reaction: Thaw the Vector and Insert (provided with the kit) on
ice. Set up the ligation reaction by the mixing the following components in the given
order:
Component Quantity
Water 7 μl
2X assay buffer 10 μl
Vector DNA 1 μl
Insert 1 μl
Ligase 1 μl
Total 20 μl
Mix the components well, Incubate at 4degC overnight. After incubation, chill the tube on
ice.
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Day 2: A. Preparation of Competent Cells

1. Inoculate 0.5 ml of the overnight grown culture into 50 ml LB media in a 250 ml conical
flask) and incubate in a 37degC shaker @ 150 rpm. Grow until the OD600 reaches 0.3.
This takes 2-3 hours.
2. Chill the culture flask on ice for 20 minutes. Transfer the 1.8 ml culture under aseptic
conditions into the sterile 2 ml vials and spin at 3500 rpm for 12 minutes in a refrigerated
centrifuge at 4degC.
3. Discard the supernatant and add 500μl of ice-cold 20mM CaCl2 to each of the vials
(placed on ice) aseptically. Suspend the cell pellets gently using a pipette. The cell pellets
must be on ice at all times of the experiment.
4. Let the vials remain on ice for 20 minutes.
5. Centrifuge the vials at 3500 rpm for 12 minutes in a refrigerated centrifuge at 4degC.
6. Discard the supernatant and re-suspend gently in 100μl of ice-cold competent cell
solution to each tube.
7. Competent cells are now ready for transformation. Transformation must be done
immediately. Do not store the competent cells.
Day 2: B. Transformation and Plating

1. Transfer a vial of competent cell onto crushed ice.
2. Transfer an aliquot of entire ligation mixture to 100 μl of the competent cells. Gently tap
the cells after addition of the DNA and place the vial back on ice immediately.
3. Incubate on ice for 30 minutes.
4. Place the competent cells in a 42degC water bath for exactly 90 seconds and quickly
place back on ice for 5 minutes. This is called the Heat-Shock step.
5. Add 900μl of LB broth under aseptic conditions to each of the vials and incubate in a
37degC shaker for 2 hours. This is called the Recovery step. This allows the bacteria to
express the antibiotic resistance gene.
6. After recovery period, spin the grown cells at 3000 rpm for 3 min at room temperature.
Decant the supernatant under aseptic conditions.
7. Add 100μl of LB broth to pellet and mix with pipette. Transfer whole 100 μl grown cells
on the LB amp plate prepared earlier. Mix well and spread thoroughly using a spreader.
Incubate the plates at 37oC overnight.
Day 3: Observations
Observe the plates for colonies. Place the plate under UV light and observe for glowing colonies.
Interpretation:
1. The antibiotic resistance is conferred on the competent cells by the plasmid, which carries
the gene for Ampicillin resistance. Hence, only the transformed colonies grow on the LB
Ampicillin plates and the untransformed cells do not grow.
2. The transformed colonies glow when exposed to UV light. The recombinant plasmid
containing active GFP gene glows under UV light.
3. Please note that the fragment cloned contained the promoter and the coding region (ORF),
from the start codon to stop codon while the vector had the terminator sequence. The host
genome produces the RNA polymerase (that recognizes the promoter) and the complete
translation machinery required for protein expression.
4. The competent cell solution is a Calcium Chloride based solution and the expected
transformation efficiency is approximately 1 X 106/μg of DNA. Lower efficiency is
attributed to improper conditions and /or bad handling of the competent cells.
5. There are colonies on plate that do not glow; those are transformants from self-ligated
vectors.
Plasmid Isolation
The DNA of prokaryotic cells (bacteria) is relatively simple in comparison to the DNA of
eukaryotic cells. A bacterium has about 1/1000 as much DNA as a eukaryotic cell. In addition to
chromosomal DNA, a bacterium may also carry an additional circular piece of DNA called a
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plasmid. Plasmids are useful to bacterial cells because they can carry genes (such as an antibiotic
resistance gene) which can allow the bacteria to grow in non ideal environments.
The plasmid has proven to be a useful tool for the molecular biologist. Genes can be inserted into
the plasmid which, in turn, can be incorporated into a bacterium by a process called
transformation. Since the bacteria will rapidly multiply and create many copies of the gene, the
molecular biologist can retrieve relatively large quantities of a protein produced by the bacteria or
can use this arrangement to study the function of a particular gene.
Principles of Plasmid Isolation

One common technique for plasmid purification is the alkaline lysis method, which breaks open
bacteria with an alkaline solution; proteins are removed by precipitation and the plasmid DNA is
recovered with alcohol precipitation. Plasmid isolation procedures are based on the fact that
plasmids usually occur in the covalently closed circular (CCC) i.e supercoiled configuration
within the host cells. After gentle cell lysis all intracellular macromolecules have to be eliminated
whereas plasmid DNA is enriched and purified. The smaller a plasmid is the easier is the isolation
of intact CCC molecules. DNA is very sensitive to mechanical stress, therefore shearing forces
caused by mixing or fast pipetting should be avoided as soon as cell lysis occurs. All mixing steps
necessary during and after cell lysis should be performed manually by inverting the tubes several
times (8-10 fold). Especially in case of larger plasmids it is recommended to cut off the very ends
of the plastic pipette tips to minimise shearing forces. Gloves should be worn in order to prevent
contamination with DNases. Autoclaved solutions, tubes and tips should be used. If phenotypic
markers of a plasmid (e.g. antibiotic resistances) are known it is advantageous to grow the cells
under selective pressure to avoid plasmid loss. If necessary, small plasmids of Escherichia coli
can easily be amplified using chloramphenicol which results in several thousand plasmid copies
per cell leading to high DNA quantities (Clewell, 1972). Large plasmids are maintained at the
level of only one copy per host cell chromosome implying higher difficulties in getting visible
DNA bands.
In general, for plasmid isolation the bacterial cultures should be grown to late logarithmic phase. It
is important to remove the supernatant completely after centrifugation from the cell pellets. Tris
buffer is the typical buffering substance for DNA having its buffering capacity in the slightly
alkaline range in which DNA can also be stored best (pH 7.5-8.2). EDTA is an important
substance in all plasmid preparations because it inhibits nuclease activity. For long-term storage,
plasmid DNA should be frozen in aliquots of storage TE buffer and repeated thawing and freezing
of DNA should be avoided.
Initiation of Culture:
1. Initiate the 5ml culture by inoculating a single colony or 500l-glycerol stock of E. coli
containing plasmid. Grow overnight at 370C at 150rpm.
2. Transfer 500l of overnight grown culture to 50 ml on LB medium with 100mg/l ampicillin
and grow overnight at 370C at 150rpm.
Plasmid Isolation:
3. Transfer the culture to two screw cap centrifuge tubes. Centrifuge at 6500rpm for 10min.
4. Discard supernatant. Resuspend the pellet in 900l of TEG buffer. Pool the pellet into one
tube. Vortex the pellet thoroughly.
5. Add 200l Lysozyme. Mix Well. Keep at room temperature for 10min.
6. Add freshly prepared 2 ml NaOH-SDS solution. Keep at RT for 10min. Mix Well.
7. Add pre-chilled 2 ml Potassium acetate solution. Keep at 40C for 10min. Mix Well.
8. Centrifuge at 10000rpm for 10min. Decant the supernatant.
9. Add two volumes of iso-propanol. Keep at 40C for at least for 60min.
10. Centrifuge at 10000rpm for 10min. Dissolve the pellet in 500l TE buffer. Collect in
eppendorf tube.
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Plasmid Purification:
11. Add 2 l RNase. Incubate on water bath at 370C for 60min.
12. Add equal volume of Phenol Chloroform (1:1). Mix well.
13. Centrifuge at 10000rpm for 10min. Take out the aqueous layer into eppendorf tube.
14. Add equal volume of Chloroform isoamyl alcohol (24:1). Mix well.
15. Centrifuge at 10000rpm for 10min. Take out the aqueous layer into eppendorf tube.
16. Add 1/10th volume of Sodium acetate and two volumes of isopropanol. Keep at 40C overnight
to precipitate the DNA.
17. Centrifuge at 10000rpm for 10min. Dissolve the pellet in 100l TE buffer.

Run 1% agarose gel as described later to visualise the isolated plasmids.
Forms of Plasmid DNA seen on Agarose Gel

Plasmids may appear in different forms: circular with different
degrees of coiling, partially cleaved or linear, and multimeric as
concatamers or catenates (doi:10.1006/abio.1999.4291). The three
forms of an uncut plasmid, all of the same size and molecular weight,
do not behave the same way during electrophoresis. Because the
Super-coiled molecules are more compact, they move more easily
through the gel, and are found farther from the point of origin in the well.
The Relaxed Circular molecules, and the Full-Length Linear molecules

usually run very close together and are not always resolved from one another.
When they do run as separate bands, either relaxed circles or linears may run
faster in the gel, depending on whether the gel is run in the presence of
ethidium bromide or not.
1. ‘Nicked Open-Circular’ DNA has one

strand cut.
2. ‘Relaxed Circular’ DNA is fully intact with
both strands uncut, but has been
enzymatically ‘relaxed’ (supercoils
removed).
3. ‘Linear’ DNA has free ends, either because
both strands have been cut, or because the DNA was linear in vivo.
4. ‘Supercoiled’ (or ‘Covalently Closed-Circular’) DNA is fully
intact with both strands uncut, and with a twist built in, resulting
in a compact form.
5. ‘Supercoiled Denatured’ DNA is like supercoiled DNA, but has
unpaired regions that make it slightly less compact; this can result
from excessive alkalinity during plasmid preparation.
1. Weigh 0.5g of agarose and add to 50 ml of 1X TAE. Boil the solution till the agarose
dissolves completely and a clear solution is seen.
2. Place the combs of the electrophoresis set in the tank such that it is 2 cm away from the
cathode. Pour the melted agarose solution into the tank without generating air bubbles.
Do not disturb the gel till it solidifies at room temperature for about an half hour.
3. Pour 200 ml of 1X TAE in the gel tank till the buffer level is seen to submerge the gel and
the electrodes. Most commonly, ethidium bromide is added to the gel (final concentration
0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After
cooling the solution to about 60C, it is poured into a casting tray containing a sample
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comb and allowed to solidify at room temperature or, if you are in a big hurry, in a
refrigerator.
4. Gently lift the combs making sure that the wells are intact.
5. After the restriction digestion reaction, add 3 μl of 6X Gel Loading Dye to each of the
reaction mix in vials. Load the entire volume of digested samples on the Agarose gel.
6. Load 20 μl of 1 kb ladder (ready-to-load) along with the digested samples.
7. Electrophorese the samples for 1 hr at 100 V.
8. Visualize the gel under UV trans-illuminator.
Several factors have important effects on the mobility of DNA fragments in agarose gels, and can
be used to your advantage in optimizing separation of DNA fragments. Chief among these factors
are:
1. Agarose Concentration: By using gels with different concentrations of agarose, one can
resolve different sizes of DNA fragments. Higher concentrations of agarose facilities
separation of small DNAs, while low agarose concentrations allow resolution of larger
DNAs. The image to the right shows migration of a set of DNA fragments in three
concentrations of agarose, all of which were in the same gel tray and electrophoresed at
the same voltage and for identical times. Notice how the larger fragments are much better
resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The
1000 bp fragment is indicated in each lane.
2. Voltage: As the voltage applied to a gel is increased, larger fragments migrate

proportionally faster those small fragments. For that reason, the best resolution of
fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to
the gel (the cm value is the distance between the two electrodes, not the length of the gel).
3. Electrophoresis Buffer: Several different buffers have been recommended for

electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-
acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat
different rates in these two buffers due to differences in ionic strength. Buffers not only
establish a pH, but provide ions to support conductivity. If you mistakenly use water
instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if
you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in
the gel to melt it.
4. Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates

between bases of nucleic acids and allows very convenient detection of DNA fragments
in gels. As described above, it can be incorporated into agarose gels, or added to samples
of DNA before loading to enable visualization of the fragments within the gel. As might
be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and
therefore its mobility.
References:
1. Ribotyping http://www.molecularepi.com/ana_ribotype.html
2. Genotyping http://en.wikipedia.org/wiki/Genotyping
3. Plasmid Isolation http://www.sci-ed-ga.org/modules/dna/plasiso.html
4. Plasmid Isolation http://www.wfcc.info/tis/info12.html
5. Agarose Gel Electrophoresis http://www.vivo.colostate.edu/hbooks/genetics/biotech
/gels/agardna.html
6. Smbrook, Maniatis and Russel (2001). Molecular Cloning, The Laboratory Manual.
Third Edition.
These pages are the property of Amit Yadav.

©2009 Amit Yadav, All rights reserved.
Comments, questions and errors should be sent to amityadav8@gmail.com

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HANDBOOK OF PRACTICAL PROTOCOLS

HANDBOOK OF PRACTICAL PROTOCOLS

Basic techniques in Genetic Engineering

Before You Start!

C. Ligation, Competent cell preparation, Transformation and Plating

E. Agarose Gel Electrophoresis

F. General Plastic ware and Equipments

Stocks and Media

8. 5M Potassium acetate (49.07gm in 100ml water). Autoclave.

9. 3M Sodium Acetate (40.83gm in 100ml water). Autoclave.

Standard Metric System of measurements: Know it well, don’t get confuse!

Restriction Fragment Length Polymorphism (RFLP)

1. They are co-dominant and unaffected by the environment;

Ribotyping (RFLP of rRNA genes) is a sensitive, highly discriminatory subtyping technique

Ribotyping is able to differentiate between different strains within a species or subspecies,

Precautions to be taken before stating experiment:

Agarose Gel Electrophoresis

Interpretation: 1. Reference Sample 1

Polymerase Chain Reaction (PCR)

Initial denaturation 94°C for 2 min

Agarose Gel Electrophoresis

Why some PCRs fail?

Why thermophilic enzymes required for PCR?

How PCR reaction cycle parameters are decided?

GENE CLONING: Ligation, Competent cell preparation, Transformation and Plating

Day 2: A. Preparation of Competent Cells

Day 2: B. Transformation and Plating

Principles of Plasmid Isolation

Agarose Gel Electrophoresis

Forms of Plasmid DNA seen on Agarose Gel

The Relaxed Circular molecules, and the Full-Length Linear molecules

1. ‘Nicked Open-Circular’ DNA has one

Agarose Gel Electrophoresis

2. Voltage: As the voltage applied to a gel is increased, larger fragments migrate

3. Electrophoresis Buffer: Several different buffers have been recommended for

4. Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates

These pages are the property of Amit Yadav.

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