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Old 04-22-2017, 01:49 PM   #1
Carcharodon
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Location: Honolulu, HI

Join Date: Jul 2015
Posts: 40
Default ddRAD: Performing PCR BEFORE Size-Selection?

Greetings!

A quick search yielded a thread asking a similar question, but it went unanswered.

Up-front, my question is: why perform a fragment size-selection before performing PCR, and has anybody here done it the other way around (PCR before size-selection for ddRAD or a similar protocol)?

Intuitively, I understand that having fragments of the proper/desired size means potentially greater amplification efficiency at that particular size (if only 200 - 300 bp fragments are available, PCR will act only on these and not fragments of other sizes because they don't exist).

However, I'm going to perform a PippinPrep size selection, and the Y-adapters will force me to select a "larger" size fragment than what I really want, because the Y-shaped adapter will slow migration through a gel. I'm looking to collect ~ 250 - 300 bp fragments (w/o adapter), and I have yet to see a good, reliable way to select fragments of this size (do I tell the machine to select 400 bp fragments? 500 bp?).

An obvious way to get around this problem is to perform PCR first, which will rid my fragments of the Y-shaped adapter, making size-selection a lot more straightforward. And I have seen folks on this forum recommend doing just that.

So...has anyone here tried this? What are some considerations I should keep in mind if I were going to try this approach?

Many thanks,
Sean
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