Preparation of genomic DNA from gram-negative bacteria

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Materials and equipment

1.  TE buffer (10 mM Tris-Cl pH at 8.0 containing 1 mM EDTA)*
2.  10% SDS
3.  20 mg/ml proteinase K in DW (stored at –20 C)
4.  5 M NaCl*
5.  CTAB/NaCl solution (10% CTABin 0.7 M NaCl)*”
6.  Chloroform/isoamyl alcohol (24:1)
7.  Phenol/ Chloroform/isoamyl alcohol (25:24:1)
8.  Isopropanol
9.  70%ethanol
10.  3 M NaOAc (pH6.0)*
11.  RnaseA (10 mg/ml)

Caution

1.  Distilled water used in this experiment should be autoclaved.
2.  * The reagent should be autoclaved.
3.  “ If necessary, heat to 65 C to dissolve.

Procedure

1.  Inoculate a 5 ml liquid culture (e.g. LB broth) with the bacterial strain of interest.
2.  Cultivate the bacteria at 37 C overnight.
3.  Spin 1.5-3 ml of the culture in a microcentrifuge for 2 min at 10,000 rpm. And discard the supernatant.
4.  Resuspend the pellet in 567 ml TE buffer by vortex.
5.  Add 30 ml of 10% SDS and 3 ml of 20 mg/ml proteinase K.
6.  Mix thoroughly and incubate at 37 C for 1 hr.
7.  Add 100 ml of 5 M NaCl and mix thoroughly.
8.  Add 80 ml of CTAB/NaCl solution, mix thoroughly and incubate at 65 C for 10 min.
9.  Add an approximately equal volume of Chloroform/isoamyl alcohol, mix thoroughly, and spin 5 min at 12,000-15,000 rpm in a microcentrifuge.
10.  Remove aqueous, viscous supernatant to a fresh microcentrifuge tube, leaving the interface behind.
11.  Add an equal volume of Phenol/ Chloroform/isoamyl alcohol, extract thoroughly, and spin in a microcentrifuge at 12,000-15,000 rpm for 5 min.
12.  Transfer the supernatant to a fresh tube.
13.  Add 0.6 vol isopropanol to precipitate the DNA (spin in a microcentrifuge at 10,000 rpm for 5 min).
14.  Add 1 ml 70% ethanol and wash the DNA to remove CTAB.
15.  Spin in a microcentrifuge at 12,000-15,000 rpm for 5 min to repellet DNA, carefully remove the supernatant and briefly dry the pellet..
16.  Dissolve the pellet in 200 ml of TE containing 20 mg of RNase A.
17.  Incubate it at 37 C for 30 min.
18.  Add 300 ml of TE and an equal vol of Phenol/ Chloroform/isoamyl alcohol, extract thoroughly, and spin in a microcentrifuge at 12,000-15,000 rpm for 5 min).
19.  Transfer the supernatant to a fresh tube and add 50 of ml of 3 M NaOAc (pH 6.0) and 1 ml of cold absolute ethanol and leave it at –20C for 1 hr.
20.  Spin at 12,000-15,000 rpm for 5 min to repellet it, carefully remove the supernatant.
21.  Add 1 ml of cold 70% ethanol and centrifuge at 12,000-15,000 rpm for 5 min to remove the salt.
22.  Remove the supernatant and briefly dry the pellet.
23.  Redissolve the pellet in 100 ml of TE buffer and measure the concentration of DNA (A260/A280).