help! PCR troubles..

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Tue Nov 9 11:50:59 EST 1993


> I am (as yet unsuccessfully) to amplify across a 3.2 kb intron in
> human genomic DNA. I am trying formamide and glycerol additions
> (2.5% and 10%, respectively) and a hot start adding dNTPS at 70
> deg. extension time is 3 min, also tried 3 min,.  with 5 sec
> added on each successive cycle. this shouldn't be as tough
> as it's going- can anyone out there give me some hints?
> has anyone out there done a fragment this large or larger?
> thanks!
> 
You may have already done this, but you should try a number of
different Mg, Taq, template, and primer concentrations.  If you have
some sort of positive control, you can optimize your reaction
conditions

James F. George, Ph.D.
University of Alabama at Birmingham
205-934-4261 voice

txpljfg at uabcvsr.cvsr.uab.edu



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