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Old 11-07-2019, 10:37 AM   #1
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Location: New England

Join Date: Jun 2012
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Default PCR amplicon library - DNA smearing

I've been making two-step amplicon libraries for years now with very few problems. The original amplicons are tailed with partial Nextera xt adapters and then I use Phusion HF to add the rest of the adapter with 6 cycles of PCR and KAPA Pure beads for cleanup. Recently, the yield has been much lower than expected and, rather than a pretty clean peak on the Bioanalyzer, there's been a lot of high molecular weight smearing. The samples labeled SPS01-07 are after the second round of amplication while the samples labeled Pre are before. Suggestions as to what might be going on?
Screen Shot 2019-11-07 at 1.34.30 PM.png
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Old 11-07-2019, 05:24 PM   #2
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Not a good explanation, nevertheless: perhaps your stock of index primers is degrading or got contaminated? You might want to order a few fresh oligos and compare?
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