PCR fails when I scale up - (Mar/03/2009 )
I'm doing restriction analysis of SNPs and whenever I scale up from 3 or so samples in the thermal cycler to 10 or 20 the efficiency of amplification drops and often the primers fail to fire for some samples. If I then go back and do only a few of these samples that failed at a time they work without a problem. What is causing this, and what can I do to be able to do more than three rxns at a time?
Redpine on Mar 3 2009, 01:39 PM said:
It is possible that your thermal cycler is unable to change the temperature quickly enough due to the increase number of samples. Might you be using an old machine?
You might not be reaching denaturation temperature in your cycler. Try increasing the denaturation and annealing times if they are currently short.