I used 100nM and 500nM final concentration for my target gene & reference gene respestively in real time RT-PCR. May I know if this is acceptable?
No this is not acceptable. Your primer concentration is high. You must dilute the primer 10 PM. Use 1 to 2 micro liter of diluted primer.
If you have primers at 10 pMol and a reaction of 20μl, 1μl of primer gives a final concentration of 500 nM. That is an acceptable concentration, but you shouldn't go too much higher. If you are concerned about the difference between the concentrations of the primer-pairs I would say that should be judged solely on the performance of the assay, not on theoretical considerations. Look at correlation coefficients (R2) and slopes of dilution curves. Make sure they are within acceptable limits.
Guys, thanks for the advice. I think I confused all of you. I used 100nM final concentration in 20uL reaction for my target gene primer pair and 500nM final concentration for my reference gene.
I don't see any problem in using primers at different conc. as long as they don't form primer dimers and are not limiting in the reaction. But it may be beneficial to use the primers at the conc. recommended by the vendor.
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