I designed primers for traditional PCR using the program vector NTI. I’ve tried to amplify 8 genes so far and so far I’ve been successful with all but one and I don’t know why. I increased the concentration of the primers (from 1/50 – 1/10) and still nothing, not even with the positive control (which has worked with all the other PCRs). I’ve checked if it could be primer dimers and hairpins formations but the primers can’t form these. Can anyone offer any advice please? The gene I’m trying to PCR encodes the Antigen 43 protein if that’s any help.
What I usually do:
-try a gradient of annealing temperature (In this case I would rather see what can be done with lower temperature)
-try a gradient of MgCl2 concentrations
-try some adjuvants (DMSO, gelatin)
Cat, yes I decreased the annealing temperature from 55-50 and it still didn’t work. I’ll suggest increasing the MgCl2 concentration to my supervisor Canalon, thanks for that advice. Mith I already run 30 cycles and I read that is the maximum under standard conditions.
Designing primers sounds fun but how do you do it? I guess you find out the flanking sequences of the gene and use these to make the primer? How do you make the primer? Where do you find the sequence information in desiging the primer?
What is your positive control? How are you evaluating a successful PCR reaction?