HELP: inconsistent genomic PCR troubleshooting

bmay bmay at 130.75.7.8
Fri Jul 28 13:41:51 EST 1995


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
Dept. Clinical Endocrinology
Medizinische Hochschule Hannover
Hannover
Germany
e-mail: ndxdbmay at rrzn-user.uni-hannover.de



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