Extraction of genomic DNA from whole blood

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 Na₂ EDTA
• 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

1. 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.
2. Spin at 3500 rpm for 15 minutes at 4^o C. 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 4^o C. The pellet should be white to cream in colour. If pellet is significantly red, repeat washing step again.
3. 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.
4. Leave to incubate for two hours at 55^o C 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.
5. Spin at 4500 rpm for 15-20 minutes at 4^o C. Pour off supernatant into a fresh tube. Take care not to dislodge pellet. Add an equal volume of cold isopropanol (stored at -20^o C). Invert 5-6 times gently to precipitate DNA.
6. 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 37^o C. 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 -70^o C.
   

References

1. Helms, C. Salting out Procedure for Human DNA extraction. In The Donis-Keller Lab - Lab Manual Homepage [online]. 24 April 1990. [cited 19 November 2002; 11:09 EST]. Available from: http://hdklab.wustl.edu/lab_manual/dna/dna2.html .
2. Epplen, J.E., and T. Lubjuhn. 1999. DNA profiling and DNA fingerprinting. Birhkhauser Verlag, Berlin. p.55.