PCR problems using Streptomyces cosmid DNA as template?

Chris Fields cjfields at jove.acs.unt.edu
Tue Feb 29 09:16:56 EST 2000


I got a PCR to work from Mycobacterium tuberculosis using Pfu polymerase
and 5% DMSO.  The results were iffy (lots of nonspecific products) when
DMSO wasn't added, so it definitely helps.  We are planning on taking
the same route with a Streptomyces cosmid.

CJF

Colin Dolphin wrote:
> 
> Dear All
> 
> I'm attempting to amplify a predicted gene in Streptomyces coelicolor
> using the appropriate cosmid (approx. 30k) DNA, containing the gene
> sequence, as template without, so far, any success. I was wondering
> whether this could be due to the high GC content of the template. Would
> fragmenting the cosmid with an appropriate RE help and/or the use of an
> additive such as DMSO?
> 
> Colin Dolphin
> Dept of Pharmacy
> Kings College London
> London SE1 8WA
> colin.dolphin at kcl.ac.uk

-- 
C. J. Fields
Graduate Student, Dept. of Biological Sciences
The University of North Texas
Denton, TX 

email : cjfields at jove.acs.unt.edu
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