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PCR fails when I scale up


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#1 Redpine

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Posted 03 March 2009 - 01:39 PM

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?

#2 perneseblue

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Posted 03 March 2009 - 03:58 PM

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?
May your PCR products be long, your protocols short and your boss on holiday

#3 phage434

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Posted 03 March 2009 - 07:11 PM

You might not be reaching denaturation temperature in your cycler. Try increasing the denaturation and annealing times if they are currently short.




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