PCR ghost bands
pcj1 at merhaba.cc.columbia.edu
Thu Jun 23 18:09:39 EST 1994
In article <9406232247.AA00726 at miranda.med.pitt.edu>,
Robert Preston <rapr at MED.PITT.EDU> wrote:
>Pierre, how many years lab experience do you have? Where do you get
>your primers? Have you ever checked the primers by HPLC or whatever?
>How many different primer sets has this happened with, and what were
>the sizes of the primers involved. Etc. This kind of mystery probably
>needs more data to solve.
I've been doing PCR for only a couple of years, but apart from that, I'm
an old hand. The primers are made in-house at one of two core facilities,
and the problems happen in both sets. The total now is about 100 pairs,
of which 60-70 pairs have problems, half of them severe, ie ghost
stronger than real band.
The primers are all 20 bp +/- 4, mp 60 C +/-1, and were selected with the
PRIMER program from the Whitehead. They have not been check by HPLC, and
we do not have the facilities for that, but I can kinase some with P32 and
run them on a gel.
The bands are not primer oligomers, by the way. A few have been sequenced
from purified bands cut out from gels, and they contained a partial
sequence from the target. However there are no obvious internal priming
Pierre Jelenc pcj1 at columbia.edu
Columbia University, New York
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