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Old 10-30-2012, 04:45 AM   #2
TonyBrooks
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Location: London

Join Date: Jun 2009
Posts: 298
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Why are you using Nextera if your amplicon size is 400-900? The Nextera kit will shear your DNA in addition to adding in the Illumina adapters. As your DNA is short to begin with, it's probably completely chewed up your amplicons - hence the failed run. Did you run the Bioanalyser on on the library before loading onto the MiSeq. You probably wouldn't have seen a product.
The varying Ampure volumes will size select at different points. 1.8X is the generic volume and will remove anything below 100bp. The less you put in, the higher the size exclusion, so 0.6X will remove most things below about 250bp (which would be your Nextera chewed DNA).
Redesign the PCRs to be longer (LRPCRs >5kb) to cover your genes, then Nextera prep.
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