INVERSE.PCR HELP WANTED

mic268a at vaxc.cc.monash.edu.au mic268a at vaxc.cc.monash.edu.au
Wed Jul 8 06:18:37 EST 1992


I am currently attempting to PCR a flanking region to a transposon insertion
using the inverse PCR method. I obtain a fragment of the expected size but
my yield is not as high as in conventional PCR. I have tried increasing the
amount of template but this does not seem to help much. I have also tried
to increase and decrease the annealing temperatures but at lower temperatures
I get more non-specifics and at higher temperatures I get even less product
I am using CsCl purified DNA and do my annealing at 50oC with an extension
at 72oC for 2 minutes. I am looking for an approx 2.0kb fragment.
Any helpful hints would be greatly appreciated.



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