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Old 02-21-2014, 09:45 AM   #3
thomasblomquist
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Location: Ohio

Join Date: Jul 2012
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Post-PCR cleanup of multiplex amplicon library prep is almost a given. Ampure beads, gel electrophoresis and band cut out, column exclusions, etc, are all decent options for Illumina library prep clean up and removal of primer dimers.

This is not the same thing as universal multiplex pcr library amplification which has a single primer that amplifies all products that have a common adapter attached on each end.

That being said, annealing temperature and time is key to minimization primer dimer formation, as well as any sort of off-target priming. There is a direct correlation between the specificity of the priming and annealing temperature. Conversely there is an inverse relationship with cycle-to-cycle yield/efficiency. Another consideration is to back off of primer concentration if specific primers are giving non-specific dimerization products.

Remember, PCR is a COMPLEX enzymatic reaction, with a lot of potential side reactions if you don't optimize the kinetics favorably for the desired product formation.

Regards,

-Tom Blomquist
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