Court Lab / Kristina Cvitanovic
Created April 2006
Salt Precipitation Method for DNA Extraction from Whole Blood
- Adapted from method found at http://www.protocol-online.org
Reagents
Buffer A (Red blood cell lysis buffer) composition (for 250 ml solution)
 0.32 M sucrose (27.38 g)
 10 mM Tris HCl (0.394 g)
 5 mM MgCl2 (0.254 g)
 0.75% Triton-X-100 (1.875 ml)
Adjust pH to 7.6
Buffer B (Proteinase K buffer) composition (for a 250 ml solution)
 20 mM Tris-HCl (0.788 g)
 4 mM Na2EDTA (0.372 g)
 100 mM NaCl (1.461 g)
Adjust pH to 7.4
*All solutions should be sterile (0.2μM filtered)
Protocol:
1. Add 2 ml of Buffer A to 2 ml of whole blood and 2 ml of cold, sterile, deionised
water. Invert tube 6-8 times and leave to incubate on ice for 2-3 minutes.
2. Centrifuge at 3500 rpm for 15 minutes. Discard supernatant into 2.5% bleach
solution. Re-suspend pellet in 2 ml of Buffer A and 6 ml of water by vortexing for 30
seconds at medium speed. Centrifuge at 3500 rpm for 15 minutes and repeat washing
step.
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 fresh, refrigerated
Proteinase K solution (20 mg/ml).
4. Leave to incubate for 2 hours at 55˚C. Remove samples and leave for 2-3 min on ice
to cool. Add 4 ml of 5.3 M NaCl (77.43 g for 250 ml solution). Vortex for 15
seconds.
5. Centrifuge samples at 4500 rpm for 20 minutes. Pour off supernatant into a fresh tube
taking care not to dislodge the pellet. Add an equal volume of cold isopropanol
(stored at - 20˚C). Invert 5-6 times gently to precipitate DNA.
6. Remove DNA with a wide bore tip and transfer to a microcentrifuge tube. Wash with
1 ml of 70% ethanol. Leave DNA to dry at room temperature. Re-suspend DNA in
300 μl of water. Allow DNA to re-dissolve overnight at room temperature.