E. coli genomic preps

Yves Brun ybrun at bio.indiana.edu
Thu Nov 11 18:57:17 EST 1993


In article <2btumd$bc7 at news.bu.edu> Patricia Foster, pfoster at bu.edu
writes:
>Dear Netters,
>     What with all the discussion of DNA purification
>kits, I thought I would ask if anyone knows of a quick
>prep for E. coli genomic DNA.  We have tried the CTAB
>protocol in Current Protocols with little success.
>I want to both clone and PCR for sequencing.  So far
>I am unhappy with results.  Any hints would be appreciated.
>Thanks
>pat
>--
>Patricia L. Foster
>Boston University School of Medicine
>Boston, MA USA
>pfoster at bu.edu

	For Caulobacter (Gram-negative, so this should also work for E. coli), I
use the following procedure which is a modification of the Magic
mini-prep from Promega (should also work with the Silica method posted on
the net recently).  It is pretty fast and gives very clean DNA. I have
successfully used this DNA for inverse PCR and Southerns.


- Isolate partially purified chromosomal DNA using a modification of the
Magic MiniPreps DNA Purification System from Promega (see attached) as
follows.
- Spin down 500 ul of an overnight culture in a 1.5 ml microfuge tube.
- Resuspend in 200 ul of 
				Tris-HCl 10 mM, pH 8.0
				EDTA 20 mM
				Sarkosyl 1%
				with 5 ul of RNaseA 10 mg/ml
- Incubate at 65?C for 10 minutes.
- Add 1ul of 20 mg/ml proteinase K and incubate at 65?C for 30 min.  to 1
hour.
- Add 30 ul ammonium acetate 7.5 M.
- Add 1 ml resin from the kit (see step V from the Promega instructions).
 Add to column, elute, wash and dry according to manufacturer's
instructions.
- Elute the DNA twice with 50 ul 65?C TE.
- Estimate the yield of DNA by running samples on an agarose gel.
______________

Yves Brun				|	e-mail: ybrun at bio.indiana.edu 
Department of Biology		|	phone: 812-855-8860
Jordan Hall 142			|	Fax: 812-855-6705
Indiana University			|
Bloomington, IN 47405-6801.	|



More information about the Methods mailing list