Thursday, September 20, 2012

Total DNA Extraction Protocol

Procedure:
1. Clean specimen and obtain a small sample for DNA extraction and place it in a sterile centrifuge tube.
2. Add 600μl nucleolysis solution to the sample and grind using a motorized pestle till the tissue degenerates. 
3. Incubate at 65°C for 15 minutes.
4. Add 3μl of RNase enzyme to break down the RNA present in the sample. Now place it at 37°C for for 15 minutes. 
5. Now add 200μl of a protein precipitation solution to the sample to precipitate all the protein and other debris present in the sample. Once added, the centrifuge tube is placed in ice for 4-5 minutes and then centrifuged for 4 minutes at maximum speed.
6. All the debris from the sample is precipitated while the DNA remains in the supernatant. 600μl of the supernatant is now removed carefully and transferred into a sterile centrifuge tube.
7. Add 600μl Isopropanol alcohol slowly to supernatant and invert in several time for the DNA to precipitate.
8. The tube is now centrifuged for 1 minute at maximum speed to completely precipitate the DNA.
9. The DNA is precipitated as the pellet and the alcohol remains in the supernatant. 
10. Remove the supernatant and add 600μl of ethanol to clean the DNA. Mix and centrifuge to precipitate DNA. 
11. Remove supernatant and air dry the DNA precipitate for 10-20 minutes.
12. Add 100μl of DNA rehydrating solution to bring the DNA into solution. Mix and place it in a water bath  at 65°for 45 - 60 minutes.
 The DNA sample thus extracted can be cooled for use in PCR or can be stored at -20°C for future use.

1 comment:

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