I have to test different primers concentration for Arabidopsis thaliana genes and its mutant by RT-qPCR. Used primers concentrations are 8µM, 10µM 12µM and 14µM. For template I used 2µL of cDNA pool (20ng).
I got Cp values in range from 28 to 34. and for some genes I got multiple (very messy) melting peaks.
Second time I tried to increase the volume of cDNA and I used 4µL instead of 2µL. Bust still the same problem occurs. The Cp values are high 29, 35, 40 and some genes have several higher melting peaks.
One explanation for high CP values is that concentration of my cDNA pool is very low.
Before running RT-PCR I checked my primers on gel and they had single amplicon. I used same temperature as for running normal qPCR. Why I am getting multiple melting peaks?
Is it possible to use (test) lower concentration of Primers (less than 8µM) to get lower CP values and better melting curves?
Thank you very much for your help!
Sincerely,
Sladja
Please re-write your question so that you indicate the final concentration of primers in your PCR reaction. Typical qPCR primer concentrations are 100-400 nM final concentration. 8uM final concentration primers would be would be way to much primers and would often result in some primer dimer formation.
Many factors can give you large Cp values: non-exponential amplification, small starting template.
Melt curves with multiple peaks are caused by multiple products. Running a gel of your PCR product can tell you if your reaction is specifically only generating the correct product.
I agree the concentration of primer written is most likely the stock, but did you use the cDNA straight ?? It has to be diluted at least 10 fold, RT-mix is a PCR inhibitor which may lead to articfact.
- Badges
- Science method