HELP: inconsistent genomic PCR troubleshooting

Ted M. tedm at darkwing.uoregon.edu
Sun Jul 30 23:26:56 EST 1995


In article <1995Jul28.184151.1540 at newsserver.rrzn.uni-hannover.de>,
bmay at 130.75.7.8 (bmay) wrote:

> 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. 100µl reaction volumes contain 200 µM dNTPs each, 1.5 mM MgCL2,
primers 50
> pmoles each, 1 µg 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 !!!
> 
> Thanks a lot in advance.
> 
> Bernhard Mayr

Welcome to PCR hell Bernhard,
   I had occasion to be there for a few months myself. A few tips I stumbled on:
   - tiny (10ul) aliquots of dNTPS, primers and buffer all made in large
batches, checked and stored at -70°C. Primers replaced at first odd
result.
  Since you don't have contamination problems you are not at the lowest
level of hell..yet.  Make sure the reactions are all truly identical; same
tubes, same oil overlay (vol. etc) same taq enzyme (tube, not lot #) An
internal control can be a big help, I used the beta globin primers for
human genomic PCR, different sized products mean you can have a truly
internal control, with four primers if need be.
                  Good luck, 
Ted Michelini
Institute of Molecular Biology
University of Oregon



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