I want to extract DNA from buccal swabs using proteinase K and the amount of DNA should be in higher concentration, PCR process.
It should do your job for PCRing, I do not see any problem. I have done as small as 8 cells for PCRing. Buccal swab will have thousands of cells. Extract DNA. You need around 50 ng or less as DNA for PCRing.
Hello Mathan Raj
To extract DNA from buccal swab samples you will require lysis buffer whose composition is as follows: [10 mM Tris (pH 8.0), 10 mM EDTA, and 2.0% SDS]. You may suspend buccal swab sample in 500ul of lysis buffer. It is then followed by addition of 5–10 μl of 20 mg/ml proteinase K. You may incubate the samples for 1–3 h at 56°C. Then carry out DNA extraction using phenol:chloroform: isoamyl alcohol solution (25:24:1).
After centrifugation for 10 min at 10,000 g (4°C), collect the upper aqueous layer, and to this aqueous layer you may add double the volume of chilled isopropanol along with one-tenth volume of 3 M sodium acetate, and chill at −20°C for 1 h for precipitation. The pellet obtained is washed with 70% ethanol, centrifuged, and then air dried. The air-dried pellet is resuspended in 50 μl nuclease-free water or 1X 10 mM Tris-HCl, 1 mM EDTA, pH 7.6 (TE), buffer and frozen at −20°C or at −80°C for storage.
Buccal swab samples will provide sufficient DNA for PCR, and PCR requires only a few nanograms of DNA.
Best.
If you want more simple method of DNA extraction, I have been using for years, just 15 minutes extraction using QuickExtract DNA Extraction Solution (enclosed sheet), no purification, no DNA measurement
1. Take PCR tube, add 50ul of DNA extraction solution (enclosed sheet), throw end of your buccal swab or 2 mm size tissue in PCR tube,
2. Heat 65C, 6min followed by 95C, 5 min in the PCR,
3. Use 2 to 4ul extract from above tube for PCRing as DNA sample, 100% PCRs have worked for me
4. Unused extract you can freeze at -20C
It takes 15 minutes to get that DNA extraction.
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