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I am preparing samples using the TruSeq DNA PCR Free library kit with UDI adaptors. The PI has requested that we perform Pippin Prep post adaptor ligation to remove adaptor contamination and target a narrow size distribution. Question is... because the protocol does not include denaturation prior to the final sequencing step, the non-homologous Y-shaped adaptors results in an atypical migration pattern on agarose gel and the Bioanalyzer results are substantially larger (750bp) than would be predicted (~350bp insert +adaptor range). Should I heat denature the samples prior to putting on Pippin Prep? Thoughts?
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Last edited by vandykitty; 01-11-2018, 08:40 AM. -
I don't have any advice but want to know if you tried this and if it worked out.Originally posted by vandykitty View Post -
The PI did not want to advert from the protocol without assurance from Illumina or Sage Science that this would work, so unfortunately, no we did not try this. Sending the samples out for sequencing today.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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