Biotechnology and Bioinformatics (1+1)

Procedure

1. Take the sample tissue (fins or muscles) and cut into small pieces with help of a pair of fine scissors. Every time wash in ethanol and cleaned with tissue paper. The fresh tissues should be homogenized.

2. Add 250 m l TEN Buffer + 20 m l 20% SDS and 10 m l Proteinase K.

TEN Buffer

100 mM Tris

10 mM EDTA pH 8.0

250 mM NaCl

Filtered 20% SDS. (add SDS to filtered water)

Proteinase K – 10 mg/ml

3. Keep the eppendorf tube containing extraction buffer/proteinase K and sample for digestion in a water bath at 55°C. The period depends upon the completion of digestion. It can be extended up to 3 days for frozen old samples. If tissue sample was more and incomplete digestion was observed some amount of Proteinase K can be (10 m l) added. For fresh tissue 3-6 hr of proteinase K digestion is enough.

4. After Proteinase K digestion take the eppendorfs from the water bath to fume hood chamber and add 300 m l phenol. Shake the tube vigorously for 20s by hand; followed by gentle mixing. Invert the tubes repeatedly. Phenol removes proteins.

5. Spin at 12000 rpm for 5 min. If rpm is less then spin for more time.

6. Remove the aqueous phase (top layer) and transfer to another eppendorf tube (when spinning, aqueous phase and organic phase are formed. Nucleic acid gets into the aqueous phase at right pH). Do not disturb tissue debris at the interface.

7. Repeat the above step once again by adding 300 m l of phenol.

8. Add 300 m l of 24:1 chloroform : isoamyl alcohol to aqueous phase. Chloroform removes phenol. Shake the tube vigorously for 20s by hand; followed by gentle mixing. The DNA and chloroform mixture should be mixed gently.

9. Spin at 12,000 rpm for 3 min.

10. Remove the bottom layer containing chloroform and discard it. Come out from the fume hood chamber.

11. Add 200 m l of isopropanol (when the eppendorf tube was shaken, clumping may be noticed as an indication for the presence of high molecular weight DNA. If the solution is clear, the DNA got degraded and broken to small fragments. Isopropanol precipitates the DNA). Mix by rapid and abrupt invertion of the tubes 5 or 6 times.

12. After adding isopropanol keep the eppendorf tube in ice for 1 hr or overnight for precipitation.

13. Spin at 12,000 rpm for 20 min. (If no pellet was observed, continue spinning for another 10 min).

14. Pour or discard the isopropanol with help of a pipette. Collect upper aqueous phase using a wide bore pipette.

15. Wash the pellet with 500 m l of 70% ethanol by gentle mixing and allow DNA to precipitate at – 20 ° C for at least 1 hr.

16. Spin at 12,000 rpm for 10 min.

17. Decant off the 70% ethanol. Remove any ethanol if present with a micropipette

18. allow the DNA to partially dry at room temperature (5-10 min) Do not over dry.

19. Add 30 m l of autoclaved nanopure water and resuspend in water bath at 55 ° C for 1 hr. DNA can be resuspended in TE buffer (10mm Tris, 1mM EDTA, pH 8.0). Allow the DNA at least 24 hrs to dissolve fully.

20. Use 1 m l aliquots for PCR.