Protocol for the preparation of E. coli genomic DNA

1) Grow E. coli K12 overnight in two 1-liter flasks containing 200 ml of L-broth.

2) Harvest cells and suspend them in 40 ml of 50 mM Tris-HCl (pH8.0) containing 50 mM EDTA.

3) Deep-freeze cells at -80 for 30 minutes.

4) Add 4 ml of 10 mg/ml lysozyme solution in 0.25 M Tris-HCl (pH8.0) to frozen cells and then

    thaw them at room temperature with occasional mixing.

5) Leave thawed cells on ice for 45 minutes.

6) Add 8 ml of STEP (0.5% SDS, 50 mM Tris-HCl (pH8.0), 0.4 M EDTA and 1 mg/ml Proteinase K)

    and incubate at 50 for one hour.

7) Add 45 ml of TE (10 mM Tris-HCl (pH8.3) and 1 mM EDTA.)

8) Then add 96 ml of phenol/chloroform/isoamylalcohol (25:24:1, saturated with TE buffer, pH9.0)

     into the tube and gently mix by repeated inversions for 5 minutes.

9) Centrifuge at 5,000 rpm (approx. 3,000 g) at room temperature for 15 minutes.

10) Transfer the aqueous phase to a new tube.

11) Add 96 ml of chloroform/isoamylalcohol (24:1) into the tube and gently mix it up and down

      in the same way as in step 8 for 5 minutes.

12) Centrifuge at 5,000 rpm (approx. 3,000 g) at room temperature for 15 minutes.

13) Transfer the aqueous phase to a new tube.

14) Add 9 ml of 3 M Soudium Acetate (pH5.2) and 225 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 in 20 ml of TE buffer by letting it stand at 4 overnight.

18) Add 10 of 10 mg/ml RNase into the DNA solution and incubate at 37 for 30 minutes.

19) Add 20 ml of phenol/chloroform/isoamylalcohol (25:24:1, saturated with TE buffer, pH9.0)

      and mix gently for 5 minutes in the same way as the step 8.

20) Centrifuge at 10,000 rpm (approx. 6,500 g) at room temperature for 15 minutes.

21) Transfer the aqueous phase into a new tube.

22) Add 20 ml of chloroform/isoamylalcohol (24:1) and mix gently for 5 minutes.

23) Centrifuge at 10,000 rpm (approx. 6,500 g) at room temperature for 10 minutes.

24) Transfer the aqueous phase into a new tube.

25) Add 2 ml of 3 M Sodium Acetate (pH5.2) and 50 ml of 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 20 ml of TE buffer by letting it stand at 4 overnight (DNA concentration

      will be around 0.1 mg/ml.)