problem of amplification - (Apr/10/2014 )
I amplified two different genes of one bacteria at the same time using gene specific primers. One gene amplification curve is like this. The melting curve shows the amplification is not specific. For the four dilutions, only the first one give signals. The primer for this gene is from published paper.
For the other gene, everything is ok.
So, what bring this kind of problem? Thank you very much for any suggestions.
It could be a different strain from the one published earlier?
There is nothing magic about published primers. Often they are poorly designed, although I suppose they once worked, at least a little. Did you try lowering the annealing temperature?