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Old 05-31-2013, 01:40 PM   #1
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Location: Czech Republic

Join Date: Mar 2013
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Default PCR purifirication prior to amplicon sequencing - individual PCRs vs. pooled PCRs?

we plan amplicon sequencing (cyt b) of cca 100 individuals (museum samples) in a 454 Junior run. I want to perform either gel cutting or Ampure purification prior to the sequencing. I just wonder if there is some reason why to purify each PCR separately or if it is ok to perform the purification on the pool of the amplicons. Possibly, the quantification of the individual PCRs (fluorometric - Qubit) is not really precise without the purification step (i.e. the amount of primer dimers etc. which are removed by the purification may vary between the samples and thus bias the concentration estimates?). Does anyone have experience with that?
Thank you very much,
dejsha is offline   Reply With Quote
Old 05-31-2013, 03:27 PM   #2
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Location: Utah

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You're right that the presence of primer dimers will throw things off. As long as the amount of primer dimers is similar in each sample, and you can tolerate a bit more variation in the amount of data per sample, you may be fine to pool first. If it were me, though, I would do the purification first. You can do that many samples with AMPure beads pretty quickly if you have a magnet for 96-well plates.
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