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Old 08-15-2019, 04:53 PM   #2
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Location: US

Join Date: Dec 2010
Posts: 449

What type of libraries are you generating? How are you measuring concentrations?

I would suggest to avoid Phusion for complex libraries. It is fine for amplicon sequencing. Phusion has been shown to introduce comparatively strong biases according to GC content and fragment lengths. Usually, NEB Q5 or Kapa Hifi are used instead.

12 PCR cycles seems way too much, when starting out with 15 ng/ul. It could be possible that the libraries are overamplified, and thus are mostly single-stranded leading to low measurements.
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