Cosmid DNA..hard to get!

Arle Kruckeberg kruckeba at biovx1.biology.ucla.edu
Wed Sep 18 20:17:13 EST 1996


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
*************************************************



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