Is your gene GC rich? If so dna secondary structure will be a problem and you may need to use up tp 10%dmso and/or a final concentration of 1Molar betaine to straighten out the dna making it more liable to amplify. Are you getting a blurred band at less than 100bp in which case you may have a primer dimer problem which would remove primers from the reaction causing it to fail

Dear Mr Paul Rutland

Thank you for your suggestion. That is a great way. However, in the case of my gene, it is 46% GC, so I think it is not a problem, and I didn't see any bands at less than 100bp.

They might still be worth trying in case local structure stops one primer annealing at an end. It may be worth orering 2 oligos 200 bp in from each end to check that the primers that you have can produce even a small product so then should work together on the longer product. Check the snp databases to see if it is possible that the 3' end of either primer is not mismatched with the sequence and possibly use another dna template in case of a snp or other sequence change

Thank you for your recommendation Mr Paul Rutland. I will try your way and find the problem in my case

I try to amplify full-length DNA from cDNA. This is my protocol:

Step 1: RNA extraction from cells

Step 2: cDNA synthesis with oligodT and RT master mix

Step 3: PCR amplification with primers for full-length DNA

after PCR amplification, I run gel electrophoresis to check if it can be amplified or not, but I can't see the band for my DNA fragment, it only primer dimer (i've already tried to fix it with many ways).

When I run PCR amplification with qPCR primers for my gene (~200bp) the results so that my cells and cDNA have very high gene expression and the band on gel electrophoresis clearly

So what happen with my full-length gene amplification?