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Extraction of DNA from Fish Fins
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Materials Required:-

 

  • Fish fins.
  • Scalpel.
  • Forceps.
  • Mortar and pestle.
  • Centrifuge.
  • Vials.
  • Micropipette and Pipette tips.
  •  Water bath.
  •  Filter paper / Blotting paper.

 

Reagents:

 

1M Tris Cl (pH 8.0) - 2.5ml.

0.5M EDTA (pH 8.0) - 50ml.

 Pancreatic RNAase - 5mg.

Proteniase K - (Required).

 

 Tris base - 10mM.

EDTA - 1mM.

 

  •  Extraction Buffer 200ML
  • TE Buffer:
  • Phenol: Chloroform: Isoamylalcohol (50ml)- (25:24:1).
  • Chloroform: Isoamylalcohol (50Ml) - (48:2).
  • 70%, 100% Ethanol (Ice cold).
  • Sodium Acetate 3M.

 

Procedure:-

 

  1. Pluck out 20-100 mg of fish fin using a sterile scalpel and a forceps.
  2. Homogenize the fins in 600μl of extraction buffer using sterile mortar and pestle.
  3. Collect the paste into 1.5ml Micro Centrifuge Tube(MCT).
  4. Incubate at 37°C for 1 hr in a water bath.
  5. Again incubate at 55°C for 1 hour in the water bath.
  6. Centrifuge at 5000 rpm for 10 minutes.
  7. Remove the supernatant put it in a new MCT and add equal volume of Phenol: Chloroform: Isoamylalcohol (25:24:1). Mix slowly and thoroughly by repeated inversion of the MCT.
  8. Centrifuge at 12000 rpm for 10 minutes and collect the top aqueous layer in a fresh MCT. Do not disturb the intermediate layer.
  9. Add  Chloroform: Isoamylalcohol in the ratio 24:1. Mix slowly and thoroughly by inversion of the tube.
  10. Centrifuge the tube at 12000 rpm for 10 minutes.
  11. Collect the aqueous layer into a fresh MCT and add 0.1 volume of 3M sodium acetate and equal volume of ice cold ethanol (100%). Mix the solution thoroughly until the DNA pellet is obtained. (Now you can see the DNA clumps in the tube.)
  12. Incubate the MCT at -20°C for 1 hour.
  13. Take out the tube and centrifuge at 1000 rpm for 10 minutes and decant the supernatant (Ethanol).
  14. Wash the tube with 70% ethanol, by centrifuging at 1000 rpm for 10 minutes and decant the ethanol carefully without losing the pellet. (If you find difficulty use pipette for removing.)
  15. Air dry the DNA pellets at room temperature by keeping the tube opened and also blot using filter paper / blotting paper.
  16. Resuspend the pellet in 50-100μl of TE buffer or sterile triple distilled water.
  17. Add 2.0μl of RNase to 100μl of DNA solution and then keep the sample at 37°C for 2 hours. (For purification.)
  18. Electrophoresis the sample on a 0.7% of agarose gel.
  19. Estimate the DNA concentration at 260nm. (OD at 260nm corresponding to 50μg/ml of double standard DNA.) Concentration of sample=Dilution factor X Absorbance.
  20. Dilute the DNA to make the concentration between 10-100μ g/ml.
  21. Use 1μl of DNA sample for PCR(Polymerase Chain Reaction).
  22. Keep the sample at -20°C or lyophilize the sample using liquid nitrogen.

 

 

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