Unspecific PCR problem - (Oct/01/2007 )

Hi there
I am trying to clone certain genes out of cDNA.
Unfortunately I am not too flexible with my primers because the regions around my start and stop codons are rather GC rich. I usually try to design primers overlapping the start - and stop codon.
After running PCR reactions with this genespecific primer pairs on my cDNA I get unspecific bands, I already tried to modify several conditions, but that did not help. For some genes it was quite easy (with the same cDNA) but for some others its really tricky and instead of my desired gene the PCR band turn out to be ß-actin or else.
Please let me know about your experiences, tricks and techniques.
pET

Hi

1) Try using touchdown PCR to increase product specificity;

2) Check if you're not trying to amplify a product that is too large for your DNA polymerase (see Long-Range PCR);

3) play with reagent concentrations in order to favor specific products;

4) if none of these work, try designing another primer pair (someones simply don't work).

Hope it helps