Protocol Online logo
Top : Forum Archives: : Molecular Biology

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.

-nat76-

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

-timjim-

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

-kr├╝melmonster-

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

http://www.pcrlinks.com/variants/touchdown.htm

-minus-

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

-djv0022-