HELP: inconsistent genomic PCR troubleshooting

Giorgio Spagnol spagnol at galactica.it
Thu Aug 3 05:18:03 EST 1995


In article <tedm-3007952142160001 at cisco-ts5-line16.uoregon.edu> Ted M.,
tedm at darkwing.uoregon.edu writes:
In article <tedm-3007952142160001 at cisco-ts5-line16.uoregon.edu> Ted M.,
tedm at darkwing.uoregon.edu writes:
> > I'm trying to amplify 2 parts of a single copy gene from human DNA
> prepared from buffy
> > coat with Qiagens QIAamp Blood Kit. I'm using 2 pairs of primers
already
> used in published
> > literature (published annealing temp. 65!/68!) designed to yield
> fragments of ~190 and
> > ~250 bp. 100!l reaction volumes contain 200 !M dNTPs each, 1.5 mM
MgCL2,
> primers 50
> > pmoles each, 1 !g DNA and 2 u cloned Taq (USB) in buffer supplied.
> Cycles consist of 1 min
> > denaturation 95!, 1 min anealing 58! and 45s extension 72! with 5 min
> initial denaturation
> > and final extension in a Crocodile II cycler from Appligene.
> > MY PROBLEM ARE INCONSISTENT AMPLIFICATION RESULTS: EITHER I GET
NOTHING
> (LOOKS LIKE A
> > PERFECT NEGATIVE CONTROL) OR I GET SINGLE BANDS BUT I CAN'T REPRODUCE
THEM THE
> > OTHER DAY. SOME DNAS YIELD SINGLE BANDS WITH ONE PRIMER PAIR AND
NOTHING
> WITH THE
> > OTHER AND VICE VERSA. I'VE ALSO FAILED TO REAMPLIFY A SINGLE BAND I
ONCE GOT!
> > Any hints, suggestions or comments wellcome !!!

If you know the sequence (or part of it) of the gene you are trying to
amplify, try using a nested, internal primer. Since I amplify with a
nested primer I solved problems like the ones you have.
best wishes.
Giorgio.



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