- Fish fins.
- Mortar and pestle.
- Micropipette and Pipette tips.
- Water bath.
- Filter paper / Blotting paper.
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.
- Pluck out 20-100 mg of fish fin using a sterile scalpel and a forceps.
- Homogenize the fins in 600μl of extraction buffer using sterile mortar and pestle.
- Collect the paste into 1.5ml Micro Centrifuge Tube(MCT).
- Incubate at 37°C for 1 hr in a water bath.
- Again incubate at 55°C for 1 hour in the water bath.
- Centrifuge at 5000 rpm for 10 minutes.
- 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.
- Centrifuge at 12000 rpm for 10 minutes and collect the top aqueous layer in a fresh MCT. Do not disturb the intermediate layer.
- Add Chloroform: Isoamylalcohol in the ratio 24:1. Mix slowly and thoroughly by inversion of the tube.
- Centrifuge the tube at 12000 rpm for 10 minutes.
- 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.)
- Incubate the MCT at -20°C for 1 hour.
- Take out the tube and centrifuge at 1000 rpm for 10 minutes and decant the supernatant (Ethanol).
- 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.)
- Air dry the DNA pellets at room temperature by keeping the tube opened and also blot using filter paper / blotting paper.
- Resuspend the pellet in 50-100μl of TE buffer or sterile triple distilled water.
- Add 2.0μl of RNase to 100μl of DNA solution and then keep the sample at 37°C for 2 hours. (For purification.)
- Electrophoresis the sample on a 0.7% of agarose gel.
- Estimate the DNA concentration at 260nm. (OD at 260nm corresponding to 50μg/ml of double standard DNA.) Concentration of sample=Dilution factor X Absorbance.
- Dilute the DNA to make the concentration between 10-100μ g/ml.
- Use 1μl of DNA sample for PCR(Polymerase Chain Reaction).
- Keep the sample at -20°C or lyophilize the sample using liquid nitrogen.
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