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DNA EXTRACTION by Nacl

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  DNA Extraction of Blood

The first step in molecular studies was to perform DNA extraction from the collected blood samples. For DNA extraction many different methods are used like manual method and through extraction kit. The DNA extraction from these samples was performed by using manual DNA extraction.

Protocol for manual DNA extraction

                                                                            Day 1

1) The frozen blood is thaw at room temperature.

2) Take 200µl of blood in an eppendorf tube. Add 600µl of tris EDTA buffer in blood sample.

3) Mix the sample by inverting several times and then centrifuge it at 3000 rpm for 5 minutes.

4) Discard about 300 µl supernatant and break the pellet that is formed at the base.

5) After the braking of the pellet, add 800-900 µl of TE buffer (blood washin buffer). Centrifuge it again at 3000 rpm for 5 minutes.

6) Repeat the last two step 4-5 times until the pellet becomes light pink.

7) Discard the supernatant. Then resuspend the pellet in 120 µl TNE, 4 µl of 10% SDS and 5µl of proteinase k.

8) Incubate the samples overnight at 37 °C in a shaking incubator.

Day 2

1) Place the tubes on ice and add 200 µl of 6M NaCl. Shake the tube vigorously.

2) Place it again on ice for 10 minutes.

3) Centrifuge the tubes at 3000 rpm for 5 minutes to pellet down the salts and protein.

4) Decant the supernatant in another new eppendorf tube

5) Add equal amount of isopropanol and invert gently until the DNA becomes visible.

6) The tubes are kept at room temperatue for 10 minutes.

7) Centrifuge at 3000 rpm for 5 minutes.

8) Supernatant is discarded very carefully

9) Wash the DNA pellet with 300 µl of 70% ethanol. Centrifuge at 3000 rpm for 10 minutes.

10) Discard 70% ethanol carefully and air dries the DNA pellet at 37 °C in an incubator.

11) Add 30 µl low TE buffer and place the tubes at 37°C shaking incubator for overnight to dissolve the DNA.


Day 3

1) Place the tubes in a shaking water bath for one hour at 70 °C temperature.

2) Keep the tubes at room temperature to cool down.

3) Spin briefly when cool down.

4) Label the eppendrofs and store at -20 °C.

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