Upload Now
In this article we will discuss about the principle, requirements and procedure for isolation of bacterial genomic DNA.
Bacterial chromosomes are single supercoiled double stranded circular DNA molecules. Plasmid DNA has a size of 0.1 to 0.5% of the chromosome. During plasmid purification preferential recovery of circular plasmid DNA over linear chromosomal (genomic) DNA is done.
Treatments with either a base or detergent will disrupt base pairing and cause the linear chromosomal DNA to denature and separate. Because of the supercoiled form and covalently closed circular nature, plasmid DNA is unable to separate and readily reforms a correctly paired super helical structure under renaturation conditions.
In case of genomic DNA preparation, detergents and other protein denaturants that are used are in high concentration compared to those used for plasmid DNA and hence the same cannot be used for plasmid DNA.
Principle:
The cell lysis solution used contains 4M guanidium thiocyanate salt which effectively denatures protein and inhibits ribonuclease and deoxy ribonucleases.
Requirements:
1. Bacterial culture in LB broth without antibiotic.
2. Cell lysis bufFer-4(M) guanidium thiocyanate salt.
3. DNA dehydrating solution.
4. Control DNA.
5. Gel loading dye.
6. 50x TAE.
7. 1.5 ml vials.
8. Eppendorf vials.
9. Ethanol 100%, 95% and 75%.
10. Electrophoresis unit.
11. Transilluminator.
12. Microfuge.
13. Pipettes.
14. Incubator.
15. Tip.
Procedure:
1. Make cell pellets of the bacterial culture in 1.5 ml eppendorf vials and keep the tube on ice.
2. Thaw the pellet at room temperature and resuspend in 700 µl of cell lysis solution at room temperature.
3. Centrifuge at 10,000 rpm for 5 minutes at room temperature.
4. Collect 500 µl of supernatant avoiding the jelley like pellet of cell debris.
5. To the 500 µl of supernatant add 1 ml of distilled ethanol. Invert tube and mix until white strands of DNA are seen precipitating out.
6. With the help of a tip, transfer this DNA into a fresh tube or centrifuge at 12000 rpm for 5 minutes and discard supernatant.
7. Wash DNA pellet with 95% ethanol, decant. Repeat this. Give final wash with 75% ethanol and air dry for 5 minutes.
8. Add 100 µl of DNA rehydrating solution and incubate at 55°-60°C for five minutes to increase solubility of genomic DNA.
9. Centrifuge at 12000 rpm for 10 minutes (to remove insoluble material and pipette out supernatant into a fresh tube).
10. Take a 30 µl of the freshly isolated DNA along with 5 µl gel loading dye, mix and load into the gel.
11. Take a 10 µl of control DNA and eletrophorise along with isolated sample in 1% agarose gel.