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Old 11-13-2014, 05:54 PM   #5
cmbetts
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Location: Bay Area

Join Date: Jun 2012
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I'd second the two-step approach. Do a first round with a fusion primer with the specific sequence and full/partial read primer sequence followed by a second round that adds the indexes and p5/p7.

That way gives you the most flexibility, as well, because you can keep reusing the indexing primers for future experiments and can always switch around which library gets which index so you don't end up with incompatible overlap.

As for cross contamination, having separate pre and post PCR benches is critical (much more common in corporate labs), but you shouldn't need to do many PCR cycles at all to add the p5/p7 and indexes, so you really just need to make sure your first PCR product and final libraries stay the hell away from the bench where you set up the first round of PCR.
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