human genomic DNA PCR

Ron Kagan rkagan at ewald.mbi.ucla.edu
Fri Sep 16 18:57:31 EST 1994


In article <35afgq$j2k at mark.ucdavis.edu> jfh, jfhess at ucdavis.edu writes:
>hello net surfers.. 
>
>this is my first posting so please pardon any " on line " faux paus I may
>make .
> 
>I am looking for tips, suggestions and/or protocols that might help me in
>my quest to use specfic as well as slightly degenerate primers in PCR
>with human genomic DNA . I am attemping to use primers from exon sequence
>to span inton sequence. So far I have fooled around with every PCR
>parameter I could rationalize changing, i.e.  cycle times, temp., and
>concentrations of primers, input DNA, enzyme, etc.
>I have been able to get PCR product with the appropiate MW using a primer
>set that does not span a inton (I use this set as positive control),
>however, my reactions that should produce larger (~ 2.5 kb) products
>don't work.
>I also know the locations and size of several intons (from
>characterization of a partial genomic clone, isolated by screening a
>library) with in the gene of interest,  which allows me to discriminate
>between junk and real product from several primer sets. 
>Once we can reproduce larger products from characterized regions, we want
>to go for the regions we don't have. If any body out there is attemping a
>similar feat and has had success, I love to hear from you and how you did
>it. 
>  

There are a number of products on the market now designed to produce long
PCR products.  One is *Stratagene's* Taq Extender mix which allegedly can
yield >10 Kb PCR.  Another is *Perkin Elmer's* GeneAmp XL kit, which is
advertised to give >20 Kb PCR.  Since The introns of some human genes can
be quite large, I'd recommend checking out the literature on one or more
of these products and trying one of them.  
See also the 15 March 1994 PNAS USA paper by Wayne Barnes on PCR up to 35
Kb.

Good Luck,

Ron Kagan

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"My Kindness is not random, and nothing senseless was ever beautiful."

                                                 - J. Zabriskie
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Ron Kagan
rkagan at ewald.mbi.ucla.edu



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