Do you need to optimize your PCR of large amplicons? Becky shares some tips for choosing the right polymerase and cycler conditions, as well as optimal handing methods, to get the most out of those long templates!
Here are some tips for amplifying large amplicons. New technologies are emerging that are enabling the cloning of larger pieces of DNA. One of these is large amplicon PCR. Here are some hints on how to get the most from those kinds of reactions.
First of all, start with a DNA polymerase that can amplify large fragments of DNA. Our Q5® DNA polymerase can successfully amplify 10 kb amplicons from complex genomic samples and 20 kb amplicons from simpler genomic templates. For larger sizes than that, our LongAmp ® Taq DNA polymerase, a mixture of Taq DNA Polymerase and a proofreading DNA Polymerase is capable of successfully amplifying up to 30 kb even from complex genomic templates.
Modify your cycling protocols. High temperatures damage DNA. And so for your denaturation and extension steps, only use as high a temperature and for as long as is required by your experiment. Note that our LongAmp Taq DNA Polymerase actually uses a 65 degree extension temperature.
Treat your DNA gently. Vortexing and excessive pipetting can damage your DNA, even fragment it. And this does not amplify well. For 20 kb targets and above, avoid pipetting once you've added your DNA to your reaction.
And finally, avoid freeze/thaw cycles of your DNA. Your DNA stocks can be aliquoted and frozen at minus 20 degrees C while your working stocks at lower concentrations can be kept at four degrees C.
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