Hi. We've been trying to prepare whole-genome DNA libraries with the TruSeq DNA library sample prep kit. I've been sharing reagents/kits, and had to split my last library prep between an older kit and a newer one. The old kit (not expired) yielded ~0.1-2.4 nM; the newer kit yielded ≥89 nM per sample. I think the problem was either age or multiple freeze/thaw cycles for the End Repair mix. We're trying to determine if it's ok to just re-amplify the libraries with low yield by a couple extra PCR cycles (we already did 10), or if we should re-make the libraries. Our concern is whether (1) the problem -- End Repair as I suspect or something else -- could somehow bias our results and yield to different results between the libraries with the two kits, and (2) if doing >10 PCR cycles could result in undue bias.
Note we're only using 1/2 reagent volume to save $, and starting with only ~300 ng dsDNA (some fresh, some old) due to limiting material. Bioanalyzer traces show ~500 bp for the size for libraries from either kit -- which I assume(?) means that the adapters ligated and we just have low yield. (We finally fine-tuned the BluePippin preps to work, but now this is causing us to ? about 22 libraries).
Any help is greatly appreciated!!!
Best,
Hilary
Note we're only using 1/2 reagent volume to save $, and starting with only ~300 ng dsDNA (some fresh, some old) due to limiting material. Bioanalyzer traces show ~500 bp for the size for libraries from either kit -- which I assume(?) means that the adapters ligated and we just have low yield. (We finally fine-tuned the BluePippin preps to work, but now this is causing us to ? about 22 libraries).
Any help is greatly appreciated!!!
Best,
Hilary
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