Hi

You mean about after you PCR?

If you are sure that your primers have a good design, then you could try with a differetn temperature gradient and look in which temperature range you get the better amplicon.

Sometiemes each primer works better at diferent Tm than other primer pairs.

I hope I understood you and helped you.

Regards

There is a difference between "a software program said these primers meet the set parameters" and "primers performed well in actual PCR reactions".

DNA can have secondary structures, some pairs of primers just are not efficient, etc.

Gareth Rostro :

Thanks for your answer. But Tm of those "not work" primers are already predicted by primer3. For most virus and bacteria, primers work well with the predict Tm. I also pay attention to the deltaTm of primer-F and primer-R ,and no one greater than 4℃.

Katie A S Burnette

Thanks for your answer. I am writing a program like you said "a software program said these primers meet the set parameters" and primer pairs should work successfully. In this program, I tried to remove the hairpin structure by avoiding 4bp complementary region occurred in a single primer and reduce the possibility of dimer formation by non-complementary region in 3'end of primer pair. I also considered Tm, GC content and GC Clamp. But primer pairs designed by this program not always work, the target sequences were there and I don't know why.

Katie A S Burnette

The parameters I considered are from the following website

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

There could be several reasons why some of your primer pairs are not working. Here are a few possibilities:

These are just a few possibilities and it's important to systematically troubleshoot the issue by testing each factor individually.

@ Ahmad Al Khraisat

Thank you very much for the answer, It really helps me a lot.