PREPARATION OF GENOMIC DNA FROM BACTERIA

Rüveyda AKÇİN, Gebze Technical University, Turkey

AIM
Isolating the genomic DNA of Escherichia coli to clone the penicillin producing ATCC 11105 gene.

INTRODUCTION
DNA is located in the chloroplasts, EDTA inhibits DNase activity by binding
mitochondrides and nuclei in the cell. The DNA magnesium, so the DNA remains stable.
molecule is negatively charged. DNA is soluble
in water however insoluble in alcohol. DNA - 10 % (w/v) Sodium dodecyl sulfate (100 g
isolation is a process of purification of DNA SDS, H2O to 1 l)
from sample using a combination of physical
The detergent increases the permeability of the
and chemical methods. DNA isolation is used
cell wall, allowing the cell contents to release.
such as forensic medicine, fingerpriting,
taxonomy, diagnosis of diseases, investigation - 20 mg/ml proteinase K
of evolutionary links, molecular genetic
studies, gene clonning. Proteinase breaks down the proteins in the cell
and provides a homogeneous mixture.
DNA isolation occurs basically in 2 stages.
Firstly, the disintegration of the cell wall. To
- 5 M NaCI (292 g NaCI, H2O to 1 l)
obtain the DNA of the cell, the cell wall needs
to be disrupted. Shredding can be done by
It passes through the cell wall to separate DNA
physical and chemical methods. Physically the
from proteins.
cells are heated, then subjected to chemical
mixtures. Chemically it is treated with solvents
- CTAB/NaCI solution (10 % CTAB in 0,7 M
such as SDS and CTAB, and the cell wall and
NaCI (Dissolve 4,1 g NaCI in 80 ml water
membrane is disturbed. RNase is used to
and slowly add 10 g CTAB
remove RNA in cells. Secondly, resolution of
(Hexadecyltrimethyl ammoniumbromide)
DNA-Protein complex and separation from
while heating and strring. If necessary, heat
other molecules. This method is denaturation.
to 65  C to dissolve. Adjust final volume to
Phenol extraction is used.
100 ml ))
MATERİALS
CTAB is used to disrupt the cell wall.
 5 ml liquid Escherichsia coli culture
and Solution required; - Phenol/chloroform/isoamyl alcohol
(25:24:1 (v/v))
- TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM - Chloroform/isoamyl alcohol (24:1(v/v))
EDTA (Dissolve 186,1 g Na2EDTA.2H2O in
700 ml H2O, Adjust pH to 8.0 with 10 M With the phenol, proteins and DNA fragments
NaOH (50 ml), Add H2O to 1 l), NaOH (10 are separated from each other. Isoamyl alcohol
M, Dissolve 400 g NaOH in 450 ml H2O, Add provides foam inhibition and RNase
H2O to 1 l )) deactivation. Some RNA molecules (e.g, mRNA)
can be removed by phenol. The only effective
way to remove RNA is by ribonuclease.
- Isopropanol and 70 % Ethanol 13. Recover the precipitated DNA by
centrifugation in the microfuge at 14.000
They facilitate the precipitation of nucleic acids. rpm for 15 min.
14. Wash DNA with 70 % ethanol to remove
PROTOCOL excess salt and CTAB from the pellet.
1. A few days before from chromosomal DNA 15. Centrifuge in the microfuge (RT) at 14.000
isolation, inoculate a 5 ml liquid Escherichia rpm for 5 min.
coli culture. Grow in conditions 16. Remove the ethanol with care and dry the
appropriate for that strain until the culture pellet in a centrifugal evaporator for 10-20
is saturated. min.
2. Transfer 1,5 ml of this culture to a 1,5 ml 17. Resuspend the dried DNA in 50 μl dH2O. (To
eppendorf and spin 2 min in 14 000 rpm. completely remove all of the chemicals.)
3. Remove the supernatant with 18. After DNA has dissolved, determine the
micropipette. concentration by measuring the
4. Resuspend the pellet in 560 μl TE buffer by absorbance at 260 nm and check the
pipette. integrity by agarose gel electrophoresis.
5. Add 30 μl of 10 % SDS and 3 μl of 20 mg/ml (Centrifuged 995 μl dH2O and 5 μl isolated
proteinase K to give a final concentration of DNA.)
100 µg/ml proteinase K in 0,5 % SDS. Mix
RESULT
thoroughly (with vortex) and incubate 1 hr
at 37 °C. Quantitation Of DNA
6. Add 100 µl of 5 M NaCl and mix thoroughly.
How is the purity and quantity of the obtained
7. Add 80 µl of CTAB/NaCl solution. Mix
DNA determined? With spectrophotometry.
thoroughly and incubate 10 min at 65 C.
The amount and purity of DNA are determined
8. Add an equal volume of
by spectrophotometer values obtained at 260
chloroform/isoamyl alcohol (24:1) mix
and 280 nm wavelengths.
thoroughly, and spin at 14,000 rpm for 5
min in microcentrifuge. (Total stock For DNA cleaning, values A (260/280) and A
volume of chloroform/isoamyl alcohol 700 (260/230) are checked.
µl. Namely, there are 672 µl chloroform, 28
The absorbance values obtained as results of
µl isoamyl alcohol in this experiment.)
9. Remove aqueous, viscous supernatant to a the performed in the experiment are as
fresh microcentrifuge tube, leaving the follows.
interface behind. Add an equal volume of A 230 → 0.040
phenol/chloroform/isoamyl alcohol
(25:24:1) and mix well but very gently to A 260 → 0.054
avoid shearing the DNA by inverting the A 280 → 0.031
tube until the phases are completely
mixed. A 330 → 0.008
10. Spin the DNA/phenol mixture at 14,000
rpm for 5 min in microcentrifuge.
11. Transfer the upper aqueous phase to a new Nucleic Concentration
tube. (Transferred total volume 500 µl.) Acid (μg/ml) A 260 unit
12. Add 0.6 volumes of isopropanol to dsDNA 50
precipitate nucleic acids. (Must be put ssDNA 33
500x0,6=300 µl). Shake the tube back and ssRNA 40
forth until a stringy white DNA precipitate
becomes clearly visible.
Optic density is 50 μg/ml for dsDNA for all that
it is 40 μg/ml for ssDNA and RNA. For example,
the following formula is utilized for
quantification of double stranded DNA;

DNA (μg/ml) = Value of OD in 260 nm x

Dilution rate x 50

According to experiment:

Dilution rate:

995 µl dH2O 5 µl DNA

1000/5 = 200 µl

DNA (μg/ml) = 0.054 x 200 x 50

= 540 μg/ml concentration of DNA

CONCLUSIONS
DNA isolation was performed to obtain the
genomic DNA of Escherichia coli. The values of
230 nm, 260 nm, 280 nm and 330 nm should be
checked to know if the isolation is clean. DNA
260, protein 280 and carbohydrates also peak
at 230 nm (maximum value). The ratio of A
(260/280) in a clean DNA is between 1.80 and
2.00; A (260/230) ratio is bigger than 2.00; A
(320) should be close to 0. A (260/280) value
below from 1.8 is protein contamination.
Furthermore, above A (260/280) value from 2
there is RNA contamination. Absorbance at 330
nm and higher indicates particles that
contaminates the solution and this value must
be 0 for nucleic acid samples.

According to this DNA isolation, A (260/280) →

1.74, A (260/230) → 1.34 and A (330) → 0.008
Firstly, we can say A (260/280) has little protein
contamination. Secondly, A (260/230) is quite
low according to this result the RNase may not
have worked or the working environment was
not sterile enough. Finally, value of A (330) can
said good because it is close to 0.