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Taqman based PCR
I am trying to amplify a PCR product of 120bp with a probe which has FAM at the 5' and BHQ1 at the 3' end. I am using the ABI Taqman Universal Master mix No amp No UNG for the amplification.
I use positive controls of cDNA for the real time. The temp used are 95 for 10 mins, 95 for 1 min, 48 for 40sec, and 60 for 1 min. 40 cycles of this. Data collection is done during the 60C cycle.
I have not been getting good results. The multicomponent data shows that there is more ROX signal than FAM signal and most of the samples positive by block based PCR and not positive during real time PCR.
At the end of the real time all the samples are run on a 1.5% agarose gel and strong bands are seen of the expected size. I am baffled as to what is going wrong.
The only thing I can suspect is the probe that I ordered, it could have degraded or not produced properly. How do I go about confirming that the probe is stable and has not been degraded.
|based , pcr , taqman|
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