Rapid & Clean Bacterial DNA
This is an adaptation of a method of Kate Wilson (Australian Inst. Marine Sci.)
suggested by Knut Jahreis (University of Osnabrück) with some modifications
by him and me.
- Pellet 1.5 mL fresh bacterial overnight culture.
- Resuspend in 567 µL TE.
- Add 15 µL 20% SDS and 3 µL 20 mg/mL proteinase K.
- 1 hr x 37°C.
- Add 100 µL 5M NaCl & mix well.
- Add 80 µL CTAB/NaCl.
- 10' x 65°C.
- Add 1 vol. CIA (~750 µL) & mix well.
- 5' spin full-speed in microcentrifuge.
- Collect aqueous phase.
- Optional: fragment DNA by repeatedly drawing through a 22 guage needle (useful when fragments do not need to be larger than ~20 KB).
- Add 1 vol. phenol/CIA (or 1/2 vol. phenol + 1/2 vol. CIA) & mix well.
- Repeat the phenol extraction once.
- Extract once with 1 vol. 1-butanol or iso-butanol.
- Add 0.6 vol. isopropanol.
- Mix well by continuous inversion.
- 5' full-speed in microcentrifuge.
- Wash pellet once with 70% ethanol - it may be necessary to spin again, as isopropanol pellets don't stick well to plastic.
- Pour off or aspirate the liquid, taking care not to lose the pellet.
- Place under vacuum no longer than 5' - there will still be some H2O left, but very little ethanol; this makes it easy to dissolve the pellet.
- Resuspend in 100 µL H2O and store at -20°C.
- (If you prefer storing at 4°C, then resuspend in 100 µL TE).
10 mM Tris [pH 7.4]
1 mM EDTA
Dissolve 4.1 g NaCl in 80 mL H2O
Slowly add with stirring 10 g
hexadecyltrimthylammonium bromide (CTAB);
— some heat may be necessary.
Adjust to 100 mL with H2O.
chloroform/isoamyl alcohol (24:1)
phenol/chloroform/isoamyl alcohol (25:24:1)
Wilson, K., "Preparation of Genomic DNA from Bacteria" in Current Protocols in Molecular Biology (1997) 2.4.1-2.4.5, Supplement 27 (Eds. F.M. Ausubel et al; Wiley InterScience)