PCR efficiency in real timePCR
Posted 11 February 2010 - 11:18 AM
Posted 16 February 2010 - 12:02 PM
That is indeed strange, as the dynamic range of real-time is quite large.
I suspect that the problem you're experiencing is related to the serial dilutions: if you are doing a 2-fold dilution, you are transferring 50% of your sample to the next dilution at a time, whereas with a 10-fold dilution, only 10% is being transferred. Thus mixing might be the problem.
Make sure that the tube is very well mixed (i.e. vortexed or pipetted up/down >40 times) after each transfer....
Posted 16 February 2010 - 10:25 PM
Hi, I cannot quite explain why, with the same DNA stock sample, I am getting >90% PCR efficiency in my realtime PCR runs using 2-fold dilutions of standard samples, but <80% efficiency using 10-fold dilution series.
Have you check on the sensitivity level of your assay as well as the saturation point of your assay?
Posted 17 February 2010 - 11:04 AM
Posted 19 February 2010 - 09:22 PM