Find content
Not Telling
Hello!
I am working on telomere measurement with real time qPCR and wondered why someone should use different concentrations of forward and reverse primers? There are a lot of publications, some researchers use 9 times more reverse than forward, some 3 times more. On what does this depend on?
Would be great to get some help!
Denise
Topics I've Started
Plateau height qPCR
10 August 2012 - 12:19 PM
I am struggling with my first qPCR on a Rotorgene Q with a SensiMix SYBR No ROX Kit. While my colleages get plateaus of ~100 (mouse DNA), I only get fluorescence levels of about 35 (on raw channel). I am working with bird DNA. My amplicon is about 100 bp. My annealing temperature I optimized by a normal gradient PCR.
What is the minimum plateau level I should have on a qPCR? I did not do a standard curve yet to calculate efficiancy bcs. I was not sure if it´s worth working on that with such low fluorescence plateaus....
I already tried a reaction with a higher gain, but this didn´t change anything....
Thank you very much for your help!
On the graph you see 2 samples in triplets....
qPCR Test - Magnification.pdf 142.4K
129 downloads
What is the minimum plateau level I should have on a qPCR? I did not do a standard curve yet to calculate efficiancy bcs. I was not sure if it´s worth working on that with such low fluorescence plateaus....
I already tried a reaction with a higher gain, but this didn´t change anything....
Thank you very much for your help!
On the graph you see 2 samples in triplets....
qPCR Test - Magnification.pdf 142.4K
129 downloads
