optimising - ct versus efficiency - (Apr/17/2007 )
I'm optimising some new primer sets and I thought I'd try something different. Normally I'd use a standard curve to judge the efficiency of the reaction over several temperatures. The one with the best dynamic range/efficiency tradeoff would win.
This time I'm running over a much larger range of temperatues, but only with one sample at a time, and looking at the Ct and instantaneous efficiency to try and guage what temperature to run at. Once I pick the temperature I'll go back and use my old method to double check that the dynamic range is OK.
Two of the primers sets are getting a good correlation between higher efficiencies and lower Cts. However another is getting a lower and lower Ct as I range the annealing temperature (Tms of the primers are ~60C, I'm up to 66C and raising atm), but the efficiencies have started to go down.
My question is whether I should shoot for the lowest Ct, or the highest efficiency?
That sounds strange, since ct is a function of efficiency when initial template number stays constant and temperature changes. The lowest ct-value must have the highest amplification efficiency. Unless you are coamplifying unspecific products in some of your dilutions, which distort the slope in your standard curve. Have you checked your products on an agarose gel?