Rapid & Clean Bacterial DNA


ECK

Description

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.


Protocol

1.
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.
2.
Add 100 µL 5M NaCl & mix well.
Add 80 µL CTAB/NaCl.
10' x 65°C.
3.
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).
4.
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.
5.
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.
6.
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).

Materials

TE
	10	mM	Tris [pH 7.4]
	1	mM	EDTA

CTAB/NaCl
	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.

CIA
	chloroform/isoamyl alcohol (24:1)

Phenol/CIA
	phenol/chloroform/isoamyl alcohol (25:24:1)


References

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)

Last Update: Thursday, 19-Jun-2014 11:36:29 PDT
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Eric Kofoid eckofoid at ucdavis.edu