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Add 1 volume of buffer A to 1 volume of blood and 2 volumes of cold, sterile, distilled, deionised water. Vortex gently or invert tube 6-8 times and leave to incubate on ice for 2-3 minutes. Spin at 3500 rpm for 15 minutes at 4oC. Discard supernatant into 2.5% bleach solution and re-suspend pellet (vortex for 30 seconds at medium speed) in 2 ml of buffer A and 6 ml of water. Spin at 3500 rpm for 15 minutes at 4oC. The pellet should be white to cream in colour. If pellet is significantly red, repeat washing step again. Add 5 ml of Buffer B and 500 µl of 10% SDS to pellet. Re-suspend pellet by vortexing vigorously for 30-60 seconds. Then add 50 µl of Proteinase K solution (20mg/ml). The Proteinase K solution should be made fresh and refrigerated prior to use. Leave to incubate for two hours at 55oC in a water bath. Remove samples and leave to cool to room temperature (or leave for 2-3 minutes on ice). Add 4 ml of 5.3 M NaCl solution. Vortex gently for 15 seconds. Spin at 4500 rpm for 15-20 minutes at 4oC. Pour off supernatant into a fresh tube. Take care not to dislodge pellet. Add an equal volume of cold isopropanol (stored at -20oC). Invert 5-6 times gently to precipitate DNA. Remove DNA with a wide bore tip and transfer to a microfuge tube. Wash with 1 ml of 70% ethanol. Leave DNA to dry for 15-20 minutes at 37oC. Re-suspend in 300-400 µl of Tris HCl, pH 8.5 (not TE!). Leave to re-dissolve overnight at room temperature. DNA can be safely refrigerated for up to a year. Long-term storage may involve ethanol at -70oC.
Extraction of genomic DNA from whole blood
Reagents
Buffer A (Red blood cell lysis buffer) composition
- 0.32 M sucrose
- 10 mM Tris HCl
- 5 mM MgCl2
- 0.75% Triton-X-100
Adjust pH to 7.6
Buffer B (Proteinase K buffer) composition
- 20 mM Tris-HCl
- 4 mM Na2EDTA
- 100 mM NaCl
Adjust pH to 7.4
N.B. All solutions should be sterile. Buffer A should be autoclaved prior to addition of Triton-X-100. Sterile filtering of solutions instead of autoclaving is a better option.
Procedure
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