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PCR no longer works - no pcr product? (Feb/24/2006 )

I have many problems with PCR, i have been using the same mixtures that I have used before but the last PCRs don´t work, the primers are the same, and all conditions too.
can somome tell me something about that?
any idea will help.


primer degradation?

nuclease contamination of template?

degradation/contamination of other components?

human error?

if you could please give us some more detail, we may be able to help...what is your protocol, what are you seeing from your attempts when you run the gel...what do your controls show?


I have used the same reagents many times before, even the same controls and all work, the protocol have been used always in my lab, I think that must being human errors, maybe the manipulation.
thanks anyway.


try with fresh prepared reagents, new aliquots etc.

check your template: apply a few µl on an agarose gel and check if it has still the right size or if it has been degraded (more smaller bands visible).

if nothing of that helps and you think it depends on human errors, ask a colleague in your lab, if he/she can try for you for one time.