primer concentrations in PCR

[Research assistant]

I'm currently working on cloning the gene for CFTR, a chloride transporter found in gills, intestine and in a few other organs, but I am having trouble getting anything on a electrophoresis gel after running a PCR, and I suspect it has something to do with my PCR.

I have three sets of degenerative primers and two sets of conserved primers that I have to choose from, but I am currently using them at a low concentration (10 microM working solution). I have also tried running a dilution series on my cDNA, but with no luck

Have any of you out there attempted running a PCR with a higher concentration of primers than I am currently using? And if you have, did you shorten the annealing time? And did you leave the annealing temperature unaltered, or did you change it? And what concentration cDNA did you use?

Hope someone wants to share previous experiences with someone who did not taker a molecular education, but who is working with it now!!

[danfive]

I responded to your other post concerning primer concentrations, so I'll ignore that here.
I worked with PCR from gills, as well. They are not very difficult but you should consider that the gills may contain a lot of blood and the iron in the hemolymph/globin is a PCR inhibitor---solution to problem: cut small sections of gill (i.e. 0.1g) and soak in pure water for 20 minutes. Take your tissue and just about any DNA extraction procedure will work and give you good quality DNA from there. To be extra secure you can use a Clontech Chromaspin Column-100 to purify your DNA a step further.

PCR is a great tool. Keep posting questions and I'm sure you'll get all your primers giving you PCR products real soon.

[pluto]

10uM is good. changing the concentrations is not going to have much of an effect. The thing that will help the most is trying to optimize the annealing temperature. Hopefully you will have a PCR machine where you can run multiple samples with different annealing temperatures to quickly test this.

Otherwise, a primer redesign is in order.