PCR from genomic DNA

Regina Shaw regina at BIOTECH.UFL.EDU
Wed Sep 29 16:28:26 EST 1999


Hi,
we're doing PCR (product size is less than 500 bp) on human genomic DNA in
96well format. The template concentration and purity is very variable and
we're trying to develop a PCR mastermix that would work as consistently
(i.e. with as few failures) as possible.
Does anyone have suggestions as to what additives and/or polymerases worked
best in their experience? Redesigning primers, optimizing Mg2+, and adding
TMAC (even though the sequence is not particularly GC-rich) has helped
alot, but the yields are still inconsistent - most templates amplify, but
some don't. It's not concentration-dependent.
Any suggestions are greatly appreciated,
Thank you very much,
Regina


Regina Shaw
Recombinant Protein Expression Lab/
   Molecular Services Core
University of Florida/ICBR
Box 110-580, South Newell Drive Bldg. 62
Gainesville, FL 32611
Voice (352) 392-3304
Fax (352) 392-4441





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