Extraction of genomic DNA from whole blood

Abstract: The protocol is simple and fairly rapid.It does not require the use of organic solvents but rather utilizes salt extraction to precipitate contaminating proteins.High quality DNA is obtained suitable for immediate PCR applications.One can obtain approximately 100-200μg of DNA from 4-8 ml of fresh or frozen 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℃.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℃.The pellet should be white t℃ream 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℃ in a water bath.Remove samples and leave t℃ool 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℃.Pour off supernatant into a fresh tube.Take care not to dislodge pellet.Add an equal volume of cold isopropanol (stored at -20℃).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℃.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℃.

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: .

2.Epplen,J.E.,and T.Lubjuhn.1999.DNA profiling and DNA fingerprinting.Birhkhauser Verlag,Berlin.p.55.