standart curve and efficiency - (Jul/13/2007 )

Hi every body,

I am doing absolute quantification real-time PCR with syber green.

I have problems with the efficiency of my standart curve that is too low (slope of my curve are -3,85 approxymatively).
I read that I should do a optimisation of the primers concentrations. I work now with a concentration of 200nM.
What are the best concentrations to test? Somebody knows a protocol?

I have another question: my sample need to be all in the standart curve? It is a problem if I have sample that have Ct up or down from the point from my standart curve?

Thanks a lot for you help!!

Hi gaianne,

the otpimal primer concentration depends on the taq you are using, normally the manufacturer gives you a hint in the manual. I would try to make both, a series with different primer concentrations (50 - 500nM) and also a series of wide range dilutions of your template (up to 1:1000).