Biochemical Techniques and Instrumentation

Procedure

1. Weigh 0.5-1 g fresh tissue in a pestle and mortar and blend the tissue.
2. Add 10 ml genomic DNA extraction buffer to the tissue blend taken in 50 ml flask.
3. Incubate the flask at 37^oC for 1h.
4. Add 50 µl proteinase K (20mg/ml stock) and mix gently.
5. Incubate in 50^oC water bath for 3h and shake gently.
6. Cool and add 10 ml saturated phenol and mix gently for 10 min.
7. Centrifuge at 3000 rpm for 15 min
8. Transfer the viscous aqueous phase to a new tube using a wide-pore glass pipette.
9. Repeat phenol extraction 2 times or more.
10. Add 2 ml ammonium acetate (10M) and mix gently.
11. Add 2 volume of ethanol
12. Swirl gently and we can genomic DNA start to form the white mass.
13. Transfer genomic DNA to a new tube by using a "U" shape pipette.
14. Air dry for 5-10min to drive off ethanol and dissolve in double distilled water or TE buffer (1X).