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-
Redpine on Mar 3 2009, 01:39 PM said:
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?
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?
-perneseblue-
You might not be reaching denaturation temperature in your cycler. Try increasing the denaturation and annealing times if they are currently short.
-phage434-