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DNA Extraction from Whole Blood 29
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Extraction of DNA from Whole Blood
John M. S. Bartlett and Anne White
1. Introduction
There are many differing protocols and a large number of commercially available
kits used for the extraction of DNA from whole blood. This procedure is one we use
routinely in both research and clinical service provision and is cheap and robust. It
can also be applied to cell pellets from dispersed tissues or cell cultures (omitting
the red blood lysis step.
2. Materials
This method uses standard chemicals that can be obtained from any major supplier;
we use Sigma.
1. Waterbath set at 65°C.
2. Centrifuge tubes (15 mL; Falcon).
3. Microfuge (1.5 mL) tubes.
4. Tube roller/rotator.
5. Glass Pasteur pipets, heated to seal the end and curled to form a “loop” or “hook” for
spooling DNA.
6. EDTA (0.5 M), pH 8.0: Add 146.1 g of anhydrous EDTA to 800 mL of distilled water.
Adjust pH to 8.0 with NaOH pellets (this will require about 20 g). Make up to 1 L with
distilled water. Autoclave at 15 p.s.i. for 15 min.
7. 1 M Tris-HCl, pH 7.6: Dissolve 121.1 g of Tris base in 800 mL of distilled water. Adjust
pH with concentrated HCl (this requires about 60 mL). CAUTION: the addition of acid
produces heat. Allow mixture to cool to room temperature before finally correcting pH.
Make up to 1 L with distilled water. Autoclave at 15 p.s.i. for 15 min.
8. Reagent A: Red blood cell lysis: 0.01M Tris-HCl pH 7.4, 320 mM sucrose, 5 mM MgCl 2 ,
1% Triton X 100.
9. Add 10 mL of 1 M Tris, 109.54 g of sucrose, 0.47 g of MgCl 2 , and 10 mL of Triton X-100
to 800 mL of distilled water. Adjust pH to 8.0, and make up to 1 L with distilled water.
Autoclave at 10 p.s.i. for 10 min (see Note 1).
10. Reagent B: Cell lysis: 0.4 M Tris-HCl, 150 mM NaCl, 0.06 M EDTA, 1% sodium dodecyl
sulphate, pH 8.0. Take 400 mL of 1 M Tris (pH 7.6), 120 mL of 0.5 M EDTA (pH 8.0),
8.76 g of NaCl, and adjust pH to 8.0. Make up to 1 L with distilled water. Autoclave 15 min
at 15. p.s.i. After autoclaving, add 10 g of sodium dodecyl sulphate.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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