DNA extraction (Phenol-chloroform Method), DNA can be extracted by different methods, following is the step wise explanation of Phenol-Chloroform DNA extraction method.
Take 700 micro-liter of blood in an eppendorf tube (1.5 micro-liter) and add 700 micro-liter of solution A*. At room temperature, gently mix it for 10 minutes and then centrifuge it at 13,500rpm for 1 minute. Discard the supernatent in 70% bleach, you will observe a visible red pellet.
Resuspend this red pellet in 400 micro-liter of solution A for 1 minute (at room temperature). Centrifuge it again at 13,500 rpm. Discard the supernatent, a light pinkish pellet will be observed.
Now add 400 micro-liter of solution B**, 20 micro-liter of proteinase K and 12 micro-liter of sodium dodecyl sulphate (SDS), vortex it in order to dissolve this pellet and store it in incubator at 37°C overnight. (You can also incubate it at 56°C for two hours).
After incubation, add 500 micro-liter of solution C*** and 500 micro-liter of phenol. Then centrifuge it at 13,500 rpm for 5 minutes.
After 5 minutes, gently transfer the supernatent to another eppendorf tube with a micro-pipette. Now add 500 micro-liter of solution C to it and mix for half a minute and re-centrifuge at 13,500 rpm for 5 minutes.
Pick the supernatent, transfer it into another eppendorf tube and add 1000 micro liter of ice chilled ethanol and 55 micro liter of sodium acetate (3 molar). Mix it for 10 minutes by gently inverting the eppendorf tube as a result a white fragile thread-like DNA will be obtained. Centrifuge for 10 minutes at 13,500 rpm.
As a result of centrifugation, a white pellet will be observed, discard the supernatent. Again wash it with ethanol (70%) and centrifuge for 5 minutes at 13,500 rpm.
Discard the white solution and air-dry the white pellet ( can be done by inverting the eppendorf tube on a tissue paper for 15 to 20 minutes at room temperature). Now add 500 micro liter of Tris-EDTA and store at 4°C.
And that’s how a DNA is extracted from blood following phenol-chloroform DNA extraction method.
* Solution A:
It can be made by dissolving 2.7 g of sucrose, 0.32 g of tris-base and 0.254 g MgCl2 in 250 ml distilled water. Autoclave it and add 1%of vol/vol triton X100. It can be stored at room temperature.
** Solution B:
It can be prepared by dissolving 3.52 g of NaCl, 0.182 g tris base and 0.11 g sodium EDTA in 150 ml distilled water. It can also be store at room temperature.
* Solution C:
Solution C can be prepared by mixing 24 ml chloroform and 1 ml isoamylalcohol in 24:1.
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