PCR is positive with one set of primers, negative with another. - (Feb/22/2006 )

Hi ! I was wondering if anyone has any good suggestions on how to solve this problem.
I have two sets of primers that are supposed to work well with the organism we are studying (giardia lamblia). With most samples both primers work well (PCR is run separately for each primer pair since the conditions for the primers are very different). But for some samples, only one primerpair works (consistently it's the same primerpair that works, the one with the lowest annealing temperature). I cant seem to find a pattern as to which samples works with one/two primerpairs, sequencing the one PCR product that works does not reveal anything special, "normal" genotype samples that should work with the second primer pair just don't sometimes.. I am having some difficulty explaining to my boss why this is happening .
I always include a positive control as well as a sample I know (from previous PCRs) is going to work with the primerpair, and they are always positive.

Hope someone can tell me what is happening!

I don't know if you order your primers at a company, sometimes they just don't work.
You also tell that the primers with the lowest annealing temperature work, maybe the GC-concentration in the other primers are too high. A higher annealing temperature is not always the best. Maybe you can try to at 1.5 microliter DMSO to a total pcr-volume of 50 microliter. This might help.

well, if you get aspecific product when lowering the annealing temperature, you're primers must be ok.
I would certainly try the DMSO, it has helped me a lot already. Or a gradient PCR. I don't know what you want to do with the product (or just test something?), but from the aspecific product, you could isolate the fragment of the correct size and do a pcr on that product.
I can't tell you why they don't work. If you use genomic DNA, the coiling of the DNA has an important role. I think there is a lot of information about it on the internet, but it's always guessing.

Double check the primer sequence that is from your genomic read. Make sure it is correct. I had this happen once. Sometimes the pcr would work and sometimes not. Actually, sometimes the sequences were nice and sometimes not. come to find out, there was a C where there was supposed to be a G. Also primer design. I had to totally redesign a forward primer just to get more consistancy in amplification. Hope this helps.