Protocol for the preparation of human genomic DNA

1) Harvest HL60 cells (from 15 petri dishes for about 1.5 x 10 cells) and wash twice with 0.7% NaCl.

2) Suspend cells with 15 ml of 10 mM Tris-HCl (pH8.3) in a polypropylene centrifuge tube.

3) Add 150 of 10 mg/ml Proteinase K and 150 of 10% SDS into cell suspension for the lysis.

4) Incubate the cell and reagent mix at 60 for one hour and subsequently at 37 for additional 16 hours.

5) Add 15 ml of phenol saturated with 1 M Tris-HCl, pH8.0, into the digest and mix gently for

   15 minutes with repeated inversions of the tube.

6) Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 minutes.

7) Transfer an aqueous phase to a new tube.

8) Add 15 ml of phenol/chloroform/isoamylalcohol (25:24:1, saturated with TE buffer, pH9.0) into

    the tube and gently mix it up and down in the same way as the step 5 for 15 minutes.

9) Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 minutes.

10) Transfer an aqueous phase to a new tube.

11) Add 15 ml of chloroform/isoamylalcohol (24:1) into the tube and gently mix it up and down

     in the same way as the step 5 for 15 minutes.

12) Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 minutes.

13) Transfer an aqueous phase to a new tube.

14) Add 1.5 ml of 3 M Sodium Acetate (pH5.2) and 30 ml of 99.5% ethanol and mix gently.

15) Collect DNA around a thin glass stick and wash it with 80% ethanol.

16) Dry DNA.

17) Dissolve DNA with 10 ml of TE buffer by letting it stand at 4 overnight.

18) Add 100 of 10 mg/ml RNaseA into the DNA solution and incubate at 37 for one hour.

19) Add 10 ml of phenol/chloroform/isoamylalcohol (25:24:1) and mix gently for 5 minutes.

20) Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 minutes.

21) Transfer the aqueous phase into a new tube.

22) Add 10 ml of chloroform/isoamylalcohol (24:1) and mix gently for 5 minutes.

23) Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 minutes.

24) Transfer the aqueous phase into a new tube.

25) Add 1 ml of 3 M Sodium Acetate (pH5.2) and 20 ml of 99.5% ethanol into the tube and mix gently.

26) Collect DNA around a thin glass stick.

27) Wash DNA with 80% ethanol.

28) Dry DNA.

29) Dissolve DNA in 2 ml of TE buffer and bring DNA concentration to 0.5 mg/ml.