2018

PREPARATION OF BACTERIAL
GENOMIC DNA AND
CONCENTRATION
DETERMINATION OF DNA
Gebze Technical University
Molecular Cell Biology Laboratory Report - 1

Ebru AKHARMAN
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PREPARATION OF BACTERIAL GENOMIC DNA AND
CONCENTRATION DETERMINATION OF DNA
AIM:
The bacterial genomic DNA is prepared from the ATCC 11105 E. coli cell line. This
genomic DNA will then be used in different studies. Cloning studies will be done with the
obtained genomic DNA.
INTRODUCTION:
Genomic DNA is an essential part of the
bacterium. Deoxyribonucleic acid is a nucleic acid
that carries genetic information that all organisms
and certain viruses must have for their vital and
biologic development. The most important task of
DNA is the long-term hiding of information. It
contains the information necessary for the
construction of components of cells such as
proteins and RNA. DNA is found both in
prokaryotes and eukaryotes. In prokaryotes, DNA is
double stranded and circular and is found
throughout the cytoplasm and is called nucleoid. In
eukaryotes, DNA is located in the nucleus and in
mitochondria or chloroplasts. The DNA in the
nucleus is double stranded and linear, whereas the
DNA in mitochondria and chloroplasts is like
prokaryotic DNA, double stranded and circular.
The isolation of DNA is one of the most
commonly used procedures in many areas of
bacterial genetics, molecular biology and
biochemistry. Purified DNA is required for many
applications such as studying DNA structure and
chemistry, examining DNA-protein interactions,
carrying out DNA hybridizations, sequencing or
PCR, performing various genetic studies or gene
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cloning. The isolation of DNA from bacteria is a
relatively simple process. The organism to be used
should be grown in a favorable medium at an
optimal temperature, and should be harvested in
late log to early stationary phase for maximum
yield.
The DNA in prokaryotes is relatively free of
associated protein, but the DNA in the nucleus of
eukaryotes is associated with basic proteins, called
histones. In order to obtain a healthy DNA, we
must attention to purity of the obtained DNA.
DNA-wrapping proteins and functional proteins
that perform other functions in cells must be
removed. Proteins, RNAs and other residues are
factors that negatively affect the purity of DNA.
The procedure of genomic DNA extraction can be
divided into 4 stages:
1. A culture of bacterial cell is grown and
harvested.
2. The cells are broken open to release their
contents.
3. The cells extracted are treated to remove all
components except the DNA.
4. The resulting DNA is then controlled.

Tasks of Useful Chemicals in Bacterial
Genomic DNA Purification:
TE Buffer: TE buffer is not used to extract DNA. It is
used to dissolve DNA that has already been
extracted and precipitated, or used in DNA
extraction protocols to help maintain the stability
of that DNA during extraction. It does this last bit
by buffering the pH of the extraction to around 7.5
- 8.5, depending on the particular make of the TE
(DNA is most stable at slightly alkaline pH), and by
EDTA chelation of metal ions (generally
Magnesium, Manganese, Zinc, etc…) which would
otherwise be used to activate DNA degrading
proteins which are present during the extraction.
This EDTA can also help destabilize cell walls and
cell membranes in some cases, though SDS,
lysozyme, and other additives are far better at cell
wall and membrane disruption.
SDS: SDS which stands for 'sodium dodecyl sulfate'.
It is strong anionic detergent that can solubilize the
proteins and lipids that form the membranes. It
removes the negative ions from the protein and
destroys its confirmation. Because of loss of
confirmation the protein loses its structure. The
proteins from the cell membrane get damaged and
cell gets broken. This will help the cell membranes
and nuclear envelopes to break down and expose
the chromosomes that contain the DNA. In
addition to removing the membrane barriers, SDS
helps release the DNA from histones and other
DNA binding proteins by denaturing them.
Proteinase K: Proteinase K is an enzyme that
cleaves the peptide bond in proteins next to the
carboxyl group of hydrophobic amino acid residues
(aliphatic and aromatic). Proteinase K itself is a
protein, but it is resistant to denaturation by heat,
detergents, and chaotropic salts and will continue
to function happily in them as long as the
temperature/concentration is not too high. It is
stable (and functional) up to 65 degrees C and will
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function in temperatures down to 25 degrees C
(room temperature). It is often used at higher
temperatures (50-65 degrees C) because most
nucleases that would chew up your DNA are
denatured/inactivated at these temperatures.
NaCl: Salting-out proteins in a sample that
contains DNA (usually a blood sample) is a cheap
and highly effective step in separating non-DNA
(cell walls, cellular proteins and other unwanted
substances) molecules from DNA molecules.
Basically, it’s a procedure where one “dismantles”
the cell and precipitates (using centrifuge) parts to
obtain really pure sample of DNA with very little
contaminants. If you add enough salt into a
solution containing proteins, they will clump
together and will be easy to remove. You have to
add enough salt to saturate the solution because
the protein would first dissolve more readily, and
only after the salt concentration is high enough will
it start clumping together and precipitating.
CTAB: Cetyltrimethylammonium bromide (CTAB) is
a nonionic detergent that can precipitate nucleic
acids and acidic polysaccharides from low ionic
strength solutions. Mean while, proteins and
neutral polysaccharides remain in solution under
these conditions. In solutions of high ionic
strength, CTAB will not precipitate nucleic acids
and forms complexes with proteins. CTAB is
therefore useful for purification of nucleic acid
from organisms which produce large quantities of
polysaccharides such as plants and certain Gram-
negative bacteria.
Phenol-Chloroform: Chloroform mixed with phenol
is more efficient at denaturing proteins than either
reagent is alone. The phenol-chloroform
combination reduces the partitioning of poly(A)+
mRNA into the organic phase and reduces the
formation of insoluble RNAprotein complexes at
the interphase. Phenol retains about 10-15% of the
aqueous phase, which results in a similar loss of

RNA; chloroform prevents this retention of water
and thus improves yields. Chloroform ensures
phase separation of the two liquids because
chloroform is miscible with phenol and it has a
higher density (1.47 g/cm3) than phenol; it forces a
sharper separation of the organic and aqueous
phases thereby assisting in the removal of the
aqueous phase with minimal cross contamination
from the organic phase.
Isoamyl alcohol: Isoamyl alcohol is added along
with Phenol:chloroform to reduce foaming and
stabilize the interphase(coagulated proteins)
between the aqueous ( which has the DNA) and
organic phase (Lipids).
70% ethanol: DNA is washed with 70% ethanol to
remove some (or ideally all) of the salt from the
pellet. If water was used as the wash then DNA
would dissolve again and if 100% ethanol was used
the salt would not wash off because sodium salts
are poorly soluble in ethanol. Because precipitation
in 100% ethanol cause removal of all water
molecule from DNA and Complete
Dehydration,which make them not soluble, So we
must give 70% wash to let it retain some water
molecule when make it soluble.
MATERIAL AND METHODS:
a) TE ( Tris / EDTA ) Buffer:
10 mM Tris-HCl (pH 8.0) is mixed with 1 mM EDTA.
b) NaOH ( 10 M ):
400 g of NaOH is dissolved in 450 ml d𝐻2 𝑂. Then,
this mixter is completed to 1000 ml and used for
EDTA ( 0,5 M and pH 8.0).
c) EDTA ( 0,5 M, pH 8.0 ):
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186,1 g of 𝑁𝑎2 EDTA.2𝐻2 𝑂 is dissolved in 700 ml
𝐻2 𝑂 and this mixture is mixed with 10 M NaOH.
Then, pH of mixture is set to 8.0 and volume of
mixture is completed to 1000 ml with d𝐻2 𝑂.
d) NaCl ( 5M ):
292 g of NaCl is completed to 1000 ml with d𝐻2 𝑂.
e) CTAB / NaCl Solution ( 10% and 0,7 M NaCl ):
4,1 g of NaCl is dissolved in 80 ml d𝐻2 𝑂 and CTAB
is added slowly . Then, the volume is completed to
100 ml with d𝐻2 𝑂.
f) SDS ( 10 % ):
100 g of SDS is dissolved in 1000 ml d𝐻2 𝑂.
NOT: The experimental steps in items a, b, c, d, e, f
must be adjusted according to the amounts to be
used.
1, 5 ml is taken from the liquid bacterial culture.
This liquid bacterial culture is centrifuged 14.000
rpm for 2 minutes.
Supernatant is removed and bacterial cell is
obtained in bottom of eppendorf.
The pellet is dissolved with 567 µl of TE buffer
and this mixture is stirred with pipetting slowly.
30 µl of 10 % SDS and 3 µl of 20 mg / ml
proteinase K are added on pellet. Then, this
materials are well mixed and incubated to 37 °C for
1 hour. (Because the cell contents are dispersed, a
sticky liquid should form. )
After 1 hour, 100 µl of 5M NaCl is added in
mixture and well stirred. ( If the concentration of
NaCl is under the 0,5 M, the CTAB helps to
precipitation of nucleic acids.)
80 µl of CTAB / NaCl solution is added and well
stirred. Then, the mixture is incubated for 10
minute in 65 °C.

700 µl of chloroform / isoamyl alcohol (24:1)
is added and well stirred. The mixture is
centrifuged with 13.000 rpm for 5 minute. (A white
interface is formed after centrifugation because of
CTAB removes protein and polysaccharides.)
The fluid on the interface consisting of protein
and polysaccharide is taken to a new eppendorf
(This fluid contain bacterial DNA). Phenol-
Chloroform- Isoamyl alcohol (25:24:1) and DNA.
Then, this mixture is upterned slowly, thus
materials mix homogeneously.
The mixture of phenol-chloroform- isoamyl
alcohol-DNA is centrifuged 13.000 rpm for 5
minutes.
After centrifuj, supernatant is taken from
eppendorf tube. (500 µl of supernatant could been
taken.)
0.6 volumes of isopropanol are added for
precipitate nucleic acids. ( 500*0,6 == 500 µl of
supernatant )
Precipitated DNA is centrifuged 13.000 rpm
for 15 minutes. (To dry DNA)
To remove excess salt and CTAB from the
pellet, the DNA should be washed with 70 %
ethanol.
Then, this mixture is centrifuged 13.000 rpm
for 5 minutes.
The 70 % ethanol is removed carefully and
The pellet is dried in a centrifugal evaporator for
10-20 minutes.
Store DNA at 4 °C for short term, -20 or -80 °C
for long term storage.
The 5 µl of dried pellet is dissolved with 995
µl of d𝐻2 𝑂 and centrifuged 13.000 rpm for 15
seconds. Then, the spectrophotometer measures
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the concentration of DNA at wavelengths of 230,
260, 280, 330 nm.
RESULTS:
DNA absorbs light with a very specific wavelength:
260 nm, in the ultraviolet range. You can shine
ultraviolet light through a solution of DNA and
measure how much of the 260 nm light gets
absorbed; a higher % absorption means a higher
concentration of DNA.
It’s common to measure the amount of 280 nm
light that gets absorbed, too. Proteins and other
contaminants absorb 280 nm light, so the ratio of
260:280 gives you a measure of the purity of the
DNA.
For more accurate readings of the nucleic acid
sample of interest, dilute the sample to give
readings between 0.1 and 1.0.
For a 1-cm pathlength, the optical density at 260
nm (OD260) equals 1.0 for the following solutions:
 a 50 μg/mL solution of dsDNA
 a 33 μg/mL solution of ssDNA
 a 20-30 μg/mL solution of
oligonucleotide
 a 40 μg/mL solution of RNA
Contamination of nucleic acid solutions makes
spectrophotometric quantitation inaccurate.
Calculate the OD260/OD280 ratio for an indication of
nucleic acid purity. Pure DNA has an
OD260/OD280 ratio of ~1.8; pure RNA has an
OD260/OD280 ratio of ~2.0. Low ratios could be
caused by protein or phenol contamination. The
dissolved DNA concentration is measured.
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230 nm 260 nm 280 nm 330 nm CONCLUSIONS:
0,093 0,243 0,140 0,001
Bacteria are a constant part of our lives. They could
be as harmless as they could be useful to us.
According to this spectrophotometer results; Bacteria have two types of DNA, plasmid and
genomic DNA. The isolation of DNA from bacteria is
1) The Concentration of Bacterial Genomic DNA:
a relatively simple process. Purified DNA is
= 50 μg/mL X 0,243 X Dilution factor required for many applications such as studying
DNA structure and chemistry, examining DNA-
Dilution factor is 200 ml. protein interactions, carrying out DNA
hybridizations, sequencing or PCR, performing
various genetic studies or gene cloning. Purification
= 50 μg/mL X 0,243 X 200 of bacterial DNA is a simple but demanding
process. Long genetic materials such as genomic
= 2430 μg/mL
DNA can be fragile, so be careful. Purification can
= 2,43 mg/ml be sequenced in four steps.

2, 43 mg/ml is concentration of DNA. First, a culture of bacterial cells is grown and

harvested. Second, the cells are broken open to
2) Indication of Nucleic Acid Purity:
their contents.Third, the cells are extracted from
𝑂𝐷260 the DNA.Forth, the resulting DNA is then
=
𝑂𝐷280 controlled. The various chemistries used during
0,243 these stages. These chemistries help to obtain DNA
= safely. The only problem that occurred during the
0,140
experiment was that the white interface of the
= 1,735 protein did not work properly.
1,735 is ratio of purity of DNA. This prevented the protein from removing the
3) The amount of thiocyanate salt: supernatant containing the DNA. This may be due
to phenol deficiency or excess. Pure DNA has an
𝑂𝐷260
= OD260/OD280 ratio of ~1.8; pure RNA has an
𝑂𝐷230
OD260/OD280 ratio of ~2.0. Low ratios could be
0,243 caused by protein or phenol contamination. The
=
0,093 purity of the obtained DNA is 1,735. This number is
= 2,612 smaller than 1.8. This may indicate protein or
phenol contamination. The DNA concentration
The ratio of 2, 612 indicates the amount of according to the obtained data was 2.43 mg / ml.
thiocyanate salt. Strong absorbance around 230nm can indicate that
4) The Turbidity in The Solution: organic compounds or chaotropic salts are present
in the purified DNA. A ratio of 260nm to 230nm can
OD330 ≌ 0 help evaluate the level of salt carryover in the
purified DNA. The lower the ratio, the greater the

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amount of thiocyanate salt is present, for example.
As a guideline, the A260/A230 is best if greater
than 1.5. According to the obtained amount, this
amount is 2, 612. This number is bigger than 1.5.
This is ideal for the purity of DNA. A reading at
330nm will indicate if there is turbidity in the
solution, another indication of possible
contamination. If the 330 nm value is close to zero
DNA is clear. If we look at the experiment in
general, bad and dirty results are not obtained
even though enzymes such as RNAase are not
used.

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