Buffer A (Red blood cell lysis buffer)
- 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.
- 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
- 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.
- 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.
- Epplen, J.E., and T. Lubjuhn. 1999. DNA
profiling and DNA fingerprinting. Birhkhauser Verlag, Berlin. p.55.