When I did the PCR with a pair of primer, the primer pair worked well and I got the desired band. But afetr that when I try to repeat the same PCR reaction, for several times, I unable to get the band. I used a new set of the primer, but still, no result. Will be pleased and benifited if someone advice me

It can be everything in your reaction. Think about controls. Run in parallel reaction with same reaction mix but different template or different primers or new buffer (enzyme, ect.). Then you will know for sure where is the problem. Good luck!

Devajit said:

When I did the PCR with a pair of primer, the primer pair worked well and I got the desired band. But afetr that when I try to repeat the same PCR reaction, for several times, I unable to get the band. I used a new set of the primer, but still, no result. Will be pleased and benifited if someone advice me

If the PCR worked very well the first time to give a good band then the most likely explanation is that you have forgotten to add one of the PCR's constituents (dNTPs, primer, template, buffer, extra Mg etc) in subsequent reactions. If you are sure that you have not done this and you have used the same program settings on the same PCR machine with the same tubes then one would look at the quality of the template. Was the template plasmid DNA or cDNA? If it is was cDNA then it will be more sensitive to degradation. Did you keep the template at -20 degrees after doing the first PCR? If it was kept at +4 degrees it will have partially degraded.

You said you got no PCR band on the gel. Did your DNA marker (ladder) bands look OK? (it is possible to forget the EtBr sometimes).

If the template was cDNA, it would be a good idea to run the PCR with a plasmid containing the same sequence. This way you can test all of the other constituents and will know that it was the cDNA template that is causing the problem (if that is what you were using).

Merry Christmas!

I tried a control reaction with another pair of primer that usually give a prominent band. I got the desired band in the control but none with the said primer pair. One of my colleague also facing the same problem with one of her primer pairs. What may be the possible reason? Is there some factor that responsible for the fast degradatiopn of primer. Please let me know if any one has such problem.

Devajit said:

I tried a control reaction with another pair of primer that usually give a prominent band. I got the desired band in the control but none with the said primer pair. One of my colleague also facing the same problem with one of her primer pairs. What may be the possible reason? Is there some factor that responsible for the fast degradatiopn of primer. Please let me know if any one has such problem.

Did you try your control reaction with the same template? Only the primers were different in the reaction mix?
You can also run reaction with your primers but different plasmid which should give you positive results.
If you are sure that it is primer then it is more easy to order new pair than to find out why they degradated.

Check the quality and concentration of your template DNA on a spec (A260/A280)

It may not have survived succesive freeze thaw cycles, especially if it is first strand cDNA from an RT rather than a plasmid.

Also is your template GC rich? you could have fluked the amplification the first time, but may need to add a denaturant to get reproducible amplification.

Also consider "Touch-down PCR", where you successively decrease the annealing temperature with each cycle of PCR if your Target is difficult.

Thanks for the advice. i will try the touch-down PCR and let you know about the result. Already I got a new paie of the primer.