RT PCR - NO PCR PRODUCT (Feb/26/2007 )

I run a commercial kit of genekam (german company) for avian flu, H7. All the required reagents are included in the kit, but labeled 'tube A,B,C' etc.
the kit contains a cDNA positive control.
the PCR protocol is consistent of 2 steps: 15'' in 95oC and 60'' in 60oC.
40 CYCLES.
There was no fluo detected - not even for the positive cDNA control. The fluo remained in baseline after 40 cycles.
what is the problem? what can i do? (i have no answer from the company so far)
Just to mention that before the 40 cycles of pcr there was no Taq activation step mentioned in the product info sheet. Is this possible?

not all taq requires activation?

I would see what the company has to say; there may be a detail you're missing...but one thing to consider is degradation? did you add TE, H2O, buffer, anything to the cDNA sample when you were setting it up, that could have contained nucleases?

2nd - you may want to run a diagnostic on your machine

3rd - check on any and all reagents that you added to the kit yourself

good luck

dear aimikins, thanks for your reply!

the kit is a commercial one and it contains everything you need, except the sample. it also contains cDNA positive and negative controls. I had to add nothing, but to mix the contains of the 'tubes' provided by the manufacturer nd run the rt pcr. But it completely failed. after 40 cycles the signal was that of baseline in the begining.

I decided to try again the positive cDNA control making the following modifications:
use 5 min Taq activation at 95oC
change annealing/extension temp from 60 to 62, or 65 degrees
and finally - if the kit fails again - i am thinking of running the product in an aagarose gel to check whether it is an amplification failure, or a fluorescence problem.

what is your opinion on this?

for the Taq the company told me that it is not a hot start Taq and it needs no activation!!! I found in Applied Biosystems site that you can skip activation stepo by adding 10 extra cycles to PCR, so that Taq is activated gradually...

hmmm....I think if it takes 50 cycles to get a product, it's not good and you may be reaching too hard

I whole-heartedly agree that at this point a gel is a good idea. if it's a mechanical problem, all the plates in the world won't give you good results