Cosmid DNA..hard to get!

martin LEACH leach at bu.edu
Thu Sep 19 13:49:41 EST 1996


the best way for me has ben to:

1. grow them up in LB broth / 30ug/ml Kanamycin (my bugs wanted this)
2. perform standard miniprep but after the potassium acetate
neutralisation...phenol phenol/chlooro then chloro extract the prep to get rid
of the protein....finally ppt the dna the usual way..

gives me reasonable yields...still low though...

scale this up for midis..

Martin

Arle Kruckeberg (kruckeba at biovx1.biology.ucla.edu) wrote:
: Dear colleagues--

: I want DNA of a cosmid clone that I bought from the American Type
: Culture Collection. The cosmid was shipped in E. coli. I streaked cells
: onto a 50 mug/ml ampicillin plate.

: I have tried to prepare DNA from these cells a number of times, by a
: variety of methods. These have included methods specifically described
: for growth of host strains containing troublesome cosmids, and for
: preparation of cosmid DNA for genome project work:

: I have tried rich and minimal media, growth at 30C (as recommended by
: the ATCC), reduced ampicillin (40mug/ml), and various lysis and DNA
: purification techniques (boiling and alkaline lysis; Wizard preps from
: Promega and CsCl banding). I have put the small yield of DNA I have
: gotten into 4 different host strains, which are transformed to AmpR by
: the DNA (though the cosmid was not evident on a gel...rearrangements?).
: I've tried prepping DNA from a lawn of cells grown on LB-Amp plates.

: All to no avail.

: Can the anyone offer any suggestions? I don't need much of the DNA,
: but I do need more than I am getting!

: Thank you,
: Arle
: *************************************************
: a.k.a. Dr. Arthur L. Kruckeberg
:  Department of Biological Chemistry
: UCLA School of Medicine
: Los Angeles CA 90024
: (310) 825-8363     FAX (310) 206-5272
: e-mail kruckeba at biovx1.biology.ucla.edu
: *************************************************

--
.....          Martin Leach                Email:leach at bu.edu
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