PCR ghost bands

[Pierre Jelenc]

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
site. 

Pierre

Pierre Jelenc                        pcj1 at columbia.edu 
                                    Columbia University, New York