Multiplex PCR

[Sabine Hanelt]

High everyone,
I'm working with the multiplex PCR to amplify the 9 exons of the HPRT gene. 
One of our problems that occures from time to time is, that the larger 
fragments (4 of 8) are very thin or not visible at all. I desided that there 
has to be a contens with the nucleotides we are using, e.g. that they are to 
old or frozen and thawn too often. But seemed not to be the only reason. The 
multiplex showed better results, but not fantastic ones. I had to use double 
of the amount of these primers I had used before and then the results were 
quite good.
Does anyone know this problem that only the larger bands get lost and has 
anyone any other good idea than doubling the amount of primer.

And another question in conection with this is, how do you store your primers 
and did you make any experiences that freezing and thawing of primers very 
often reduces the quality of these primers?

Well, thank's a lot for your help
Sabine

Sabine Hanelt
Abt. Medizinische Genetik
Uni Klinik Ulm
D-89069 Ulm
Phone: +49-731-502-3432
Fax: +49-731-502-3438