Sometimes at 0.5 micromolar we observe amplification in template control. How to avoid this?
I need to know the exact concentration required.
Hello,
A good concentration in qPCR has to be optimized. The range for concentration is 0.2 to 1µM in general and your qPCR system need on optimized one. You can start trying 0.2 / 0.5 / 0.8 and 1.
Comparing this concentration on standard curve answer will help you to chose. But do not forget also that some qPCR system work better with non equivalent primer concentration. Actually, I made several optimization where forward and reverse primer had not have the same concentration, like 0.3µM for the forrward and 0.6µM for reverse one. It can happen and has to be also checked.
It is a little matrix of concentration to test and take you 2 or 3 days to do, but can lead to much better amplification then...
Good luck
Hello,
A good concentration in qPCR has to be optimized. The range for concentration is 0.2 to 1µM in general and your qPCR system need on optimized one. You can start trying 0.2 / 0.5 / 0.8 and 1.
Comparing this concentration on standard curve answer will help you to chose. But do not forget also that some qPCR system work better with non equivalent primer concentration. Actually, I made several optimization where forward and reverse primer had not have the same concentration, like 0.3µM for the forrward and 0.6µM for reverse one. It can happen and has to be also checked.
It is a little matrix of concentration to test and take you 2 or 3 days to do, but can lead to much better amplification then...
Good luck
500 nanomolar final concentration? This is relatively high, we usually used a concentration between 150 and 300 nanomolar. If you want (or have to) use high primer concentrations, you usually have to adjust the magnesium concentration as well, since DNA binds magnesium and this can lead to non-optimal PCR conditions or complete inhibition.
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