The Isolation of Genomic DNA from E. coli is explained in this section
Illustration of genomic DNA isolation
Aim of the experiment: To isolate the genomic DNA from Escherichia coli DH5α cells.
PRINCIPLE OF THE EXPERIMENT
The isolation and purification of DNA from cells is one of the most
common procedures in contemporary molecular biology and embodies a
transition from cell biology to the molecular biology (from in vivo to
in vitro). 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 genomic DNA isolation needs to separate total DNA from 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
used to disrupt the cell membrane. Once cell is disrupted, 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 FOR GENOMIC DNA ISOLATION
- LB broth
- Tris EDTA (TE) buffer
- 10 % SDS
- Proteinase K
- Phenol-chloroform mixture
- 5M sodium acetate (pH 5.2)
- 70% ethanol
- Autoclaved distilled water
- Eppendorf tubes (2 ml)
PREPARATION OF REAGENTS FOR GENOMIC DNA ISOLATION
- 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
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).
7. 70% ETHANOL
PROCEDURE FOR CARRYING OUT GENOMIC DNA ISOLATION
- 2 ml overnight culture is taken and the cells are harvested by
centrifugation for 10 minutes.
- 875 µl of TE buffer is added to the cell pellet and the cells are
resuspended in the buffer by gentle mixing.
- 100 µl of 10% SDS and 5 µl of Proteinase K are added to the cells.
- The above mixture is mixed well and incubated at 37º C for an hour in
- 1 ml of phenol-chloroform mixture is added to the contents, mixed
well by inverting and incubated at room temperature for 5 minutes.
- The contents are centrifuged at 10,000 rpm for 10 minutes at 4º C.
- The highly viscous jelly like supernatant is collected using cut tips
and is transferred to a fresh tube.
- The process is repeated once again with phenol-chloroform mixture
and the supernatant is collected in a fresh tube.
- 100 µl of 5M sodium acetate is added to the contents and is mixed
- 2 ml of isopropanol is added and mixed gently by inversion till white
strands of DNA precipitates out.
- The contents are centrifuged at 5,000 rpm for 10 minutes.
- The supernatant is removed and 1ml 70% ethanol is added.
- The above contents are centrifuged at 5,000 rpm for 10 minutes.
- After air drying for 5 minutes 200 µl of TE buffer or distilled water is
- 10 µl of DNA sample is taken and is diluted to 1 or 2 ml with distilled
- The concentration of DNA is determined using a spectrophotometer at
- The remaining samples are stored for further experiments.
PRECAUTIONS TO OBSERVE WHEN PERFORMING GENOMIC DNA ISOLATION
- Cut tips should be used so that the DNA is not subjected to
- Depending on the source of DNA the incubation period of Proteinase
K should extended.
- The phenol chloroform extraction should be repeated depending on
the source of DNA to obtain pure DNA.
- DNase free plastic wares and reagents should be used.
Results and discussion:
References: DEPARTMENT OF BIOTECHNOLOGY, SCHOOL OF BIOENGINEERING, SRM UNIVERSITY