Hi
I am following a Pharmacia protocol for mini-preps from S. aureus. It
involves lysostaphin treatment (5 ug/mL) for 1 hour at 37C, followed by
incubation with lytic solution (NaOH & SDS) at 70C for 20 min. I then
Phenol extract and Ethanol precipitate the DNA.
My problem is - When I run this DNA on agarose gels (no restrction
digests), I get a band corresponding to the plasmid I am supposed to have.
I am attributing this to breaks in dsDNA and a small fraction runs as
a linear piece. However, I get a very high amount of DNA (molecular
weight > 20 kb. When I digest the DNA sample with unique (to my plasmid)
enzymes, i get a smear or multiple bands. This tells me that the big glob
of DNA is chromosomal DNA. When I precipitate the DNA, i usually see a
big pellet.
My question - how can i modify this protocol to eliminate or minimize
chrom. DNA? I need to use this DNA for cloning (yes!!) and hence, need
some way of getting rid of the chrom. DNA
Thank you
Arul
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Arul Jayaraman
Dept. of Chemical & Biochemical Engineering
University of California, Irvine
Irvine, CA 92697-2575
Ph : (714)-509-1060 (H)
(714)-824-8389 (L)
(714)-824-2541 (Fax)
http://www.eng.uci.edu/~arul/
