Hello!

I'm trying to perform a TaqMan qPCR to check for certain fish species in environmental DNA samples. I am using a published primer + probe set. The probe is carrying FAM at it's 5' end and MGB-Q500 at its 3' end. When running the qPCR, I see no increase in the fluorescence signal. However, when I run the reaction on a gel, I do see nice PCR products of the expected size (same when I run a "conventional" PCR just with the primer pair), so I am assuming that my probe is either not annealing or something with the fluorescence signal detection is not working. I was running another TaqMan essay with similar conditions (same modification, same chemistry) on the same machine in parallel, which works fine.

Is there a way to test if the probe is annealing? I was thinking about running a PCR just with the probe as a substitute for the forward primer, but the 3' modifications would inhibit the extention anyay, right?

I'm using the Thermo Fisher Environmentam Master Mix 2.0 with the its standard cycling condions (which are also the conditions published)

Tms:

(calculated with Thermo Fisher Mutliple ologo analyser):

F-primer: 62,8°C

R-Primer: 67°C

Probe (without MGB): 63,6°C

Any help is very much appreciated! Thank you!!

Tamara

More Tamara Schenekar's questions See All
Similar questions and discussions