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Help needed. PCR band problem - (May/14/2007 )

I have designed a set of primer pairs for a particular gene. The first PCR result showed positive result for this gene but the band was weak. A second attempt on amplifying the gene with the same set of primer pairs did not generate any bands at all. Would like to know the reason why a positive result on the first attempt but a negative outcome after the second try of the PCR. Thank you.


Erm, I think you should list down your conditions first. Pretty hard to judge without knowing your temp, concentration etc. wink.gif


Are you sure that you had the correct product in the first trial?


timjim is amazingly right, ,
But if you wanna clone that later on and if we assume that you do everything well, then don't bother anymore and re-amplify using the first PCR product as template.

If you don't wanna clone but don't want to bother either wink.gif , just go for TOUCHDOWN PCR


You can try to do an agarose gel [1.5%] electrophoresis and load 400mM of your primers for check integrity (if they are degraded).