ISOLATION
OF GENOMIC DNA FROM BACTERIA
AIM: To isolate the genomic DNA from the given bacterial culture.
PRINCIPLE:
The bacteria should be
grown in Luria-Bertani medium at optimal temperature for
overnight incubation. The late log phase to early stationary phase culture
gives maximum yield of DNA due to active replication of DNA is taking place in
this stage of bacterial cell growth.
The genomic DNA not only contains total DNA but also RNA, protein,
lipid, etc. Initially the cell membranes must be disrupted in order to release
the DNA in the extraction buffer. SDS (sodium dodecyl sulphate) is disrupting
the cell membrane and then, the endogenous nucleases tend to cause extensive
hydrolysis. Nucleases apparently present on human fingertips are notorious for
causing spurious degradation of nucleic acids during purification. DNA can be
protected from endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion
is considered as a necessary cofactor for action of most of the nucleases.
Nucleoprotein interactions are disrupted with SDS, phenol or proteinase K.
Proteinase enzyme is used to degrade the proteins in the disrupted cell soup.
Phenol and chloroform are used to denature and separate proteins from DNA.
Chloroform is also a protein denaturant, which stabilizes the rather unstable
boundary between an aqueous phase and pure phenol layer. The denatured proteins
form a layer at the interface between the aqueous and the organic phases which
are removed by centrifugation. DNA released from disrupted cells is
precipitated by cold absolute ethanol or isopropanol.
MATERIALS REQUIRED:
LB Broth, TE buffer (pH 8.0), 10% SDS, Proteinase K,
Phenol-chloroform mixture, 5M Sodium Acetate (pH 5.2), Isopropanol, 70% ethanol,
Autoclaved Distilled Water, Eppendorf tubes, 2 ml Micropipette, Microtips .
PROCEDURE:
1. 10 mL of mid-
to late-log-phase culture (0.5 – 0.7 at OD600) was Transferred to a tube and
the cells were pellet out through centrifugation at 7,500 rpm for 10 minutes. The
supernatant was discarded.
2.
Pellet was Resuspended with 467 μL RNase A in TAE Buffer and transferred to a
1.5-mL microcentrifuge tube and Added 8 μL lysozyme and then incubated at 37oC
for 60 minutes.
3. 30 μL 10% SDS
(sodium dodecyl sulfate) was added and mixed with 3 μL proteinase K, which was gently
inverted and incubated at 50oC for 60 minutes.
4. 525 μL PCI (Phenol:Chloroform:Isoamyl)
solution was added and mixed for 10 minutes by gentle inversion. This set up
was centrifuged at 12,000 rpm for 15 minutes.
5.
The upper aqueous phase was transferred to a sterile 1.5-mL microcentrifuge
tube, without disturbing the bilayer.
6. An equal volume of 100% ethanol was added
and gently mixed by inversion and Centrifuged at 12,000 rpm for 20 minutes.
7.
Carefully decanted the supernatant and thoroughly dry pellet at room
temperature or in a 50oC incubator.
8. The pellet was Resuspend in 50 μL TE
(Tris-EDTA) buffer and allowed the pellet to set overnight at 4oC.
9.
Presence of bacterial DNA could be confirmed by running 5 μL of product on a
1.5% agarose gel. Purified DNA would appear as a defined band when visualized
under UV light.
Preparation of Reagents:
1.
TE BUFFER (pH 8.0): 10 mm Tris HCl (pH 8.0), 1 mm EDTA (pH 8.0)
2. 10% SDS: Dissolve 10 g of SDS in 100 ml
autoclaved distilled water.
3.
PROTEINASE K: Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled
water. 4. PHENOL – CHLOROFORM MIXTURE: The pH is very important. For RNA
purification, the pH is kept around pH 4, which retains RNA in the aqueous
phase preferentially. For DNA purification, the pH is usually 7 to 8, at which
point all nucleic acids are found in the aqueous phase. Mix equal volume of
phenol with chloroform. Keep the mixture on ice and add 20 ml TE buffer,
extract by shaking for 15 minutes. Remove the dust on the surface layer using a
pipette. Repeat 4-5 times. Add 30-40 ml of TE buffer and store it on ice.
5.
5M SODIUM ACETATE: Dissolve 41 g of sodium acetate in 100 ml distilled water and adjust pH with dilute
acetic acid (pH 5.2).
6.
ISOPROPANOL
7.
70% ETHANOL
RESULT
AND DISCUSSION:
