High background fluorescence detected before starting PCR - (Nov/02/2015 )

I would like to ask about a  problem recently noticed with taqman probes PCR.  We check PCR reactions before starting pcr on a fluorometer and we had very high  fluorescence  background upon mixing only the fam probe with the reverse primer and the polymerase+buffer. Testing separately or in couple combination  the certain reagents did not result in fluoresscence. The problem was not observed with the forward primer and the probe +polymerase. The polymerase is also thermostable and the fuorescence is noticed at room temperatures before starting PCR. The same PCR reagents( pol+buffer)  with  a hex probe but different target from fam probe  did not have any problem.  Any idea why only mixing fam probe +reverse only primer +pcr Taq polymerase results almost instantly in fluorescence at room temperature?

All the best

Jim 

May interact with the quencher, so that probe is emmiting fluorescence while intact? I don't know.

Does it happen during PCR? If not, I would not care about it.

I never heard of anyone measuring a PCR mix on fluorometr before PCR start, so who knows, maybe this is actually common with some probe and primer combinations. 

More important is, if it interferes with your fluorescent measurements during real-time, you could see differences in the baseline fluorescence in such case.

Hi Trof;

Thanks for your reply, I am on a similar line to explain the problem. The reason we do testing prior to PCR reacion is that we do end point fluoresence for a  qualitative PCR testing. If  reactions were on a Real time PCR instrument the problem may was not there as primer -probe interactions because of measuring fluoresence at higher temperatures would not exist. Our instrumentation for end point PCR permits measuring only at room temperature.  One way or another a melting curve analysis could also give us some information. May be measuring fluoresence  at room temperature the only solution is to redesign the certain primer.

I see. For endpoint you indeed need to know the background.

Could you perhaps simulate the PCR reaction by runing few cycles only (with denaturation, or increased denaturation time to simulate multiple cycles) to see if you observe background fluorescence after that (few cycles should generate only negligable amount of new DNA, below threshold)? Maybe it will disappear so then you know, it doesn't interact with your end-point measurements.

What kind of polymerase you have? Is it hot-start (not sure what you meant by "thermostable" all PCR polymerases are thermostable, maybe you meant "inactive until thermal activation" aka hot-start)? If so what kind, antibody, covalent modification... etc.?

Also, general recommendation how to minimize primer interactions is denature primers breafly and then cool quicky on ice, and ideally keep cool until reaction starts. Since it is not just primers that seem to interact (and since you do measurements at room temperature) it may not have any effect but who knows.