For 10 mL of whole blood in EDTA
1. Thaw frozen blood samples to room temperature
2. Mix blood by inversion and pour into labelled 10 mL centrifuge tubes
3. Centrifuge at ~2800 rpm for 20 minutes
4. Carefully pour off supernatant into a blood waste container, leaving pellet
5. Add 10 mL of "cell lysis buffer" (20 mM Tris, 5 mM EDTA, pH 7.5) (use approximately 7 mL if there is only 5 mL of blood)
6. Resuspend cells
7. Centrifuge at ~2800 rpm for 10 minutes
8. Carefully pour off supernatant into a blood waste container, leaving pellet
9. Repeat steps 5 - 8 twice, leaving a clean pellet of white cells
10. Resuspend the pellet in 2.0 mL of "salting out digestion buffer" (20 mM Tris, 5 mM EDTA, 400 mM NaCl). Mix thoroughly.
1. Add 150 mL 10% SDS and 150 mL 10 mg/mL Proteinase K. Mix thoroughly
2. Incubate overnight in a water bath at 45C
(Miller et al, 1988)
1. Add 0.8 mL saturated NaCl and shake vigorously
2. Centrifuge at ~2800 rpm for 10 minutes
3. Shake gently to loosen precipitate from froth
4. Centrifuge at ~2800 rpm for a further 5 minutes
5. Transfer the "clear" supernatant to a labelled centrifuge tube containing 6.5 mL 95% ethanol
6. Gently mix - the DNA will come out of solution
7. Transfer the clump of DNA to a labelled eppendorf containing 500 mL of 70% ethanol, mix, and centrifuge for two minutes
8. Remove ethanol and air dry. Add an appropriate amount (250-1000 mL) of sterile TE (10 mM Tris, 0.1 mM EDTA, pH 8.0)
9. Allow the DNA to dissolve for several days before quantitating and storing at -20C