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Old 02-26-2020, 11:13 AM   #1
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Location: CT

Join Date: Apr 2015
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Default Nextera Flex with poor DNA, reduce tagmentation time?

I have samples for shotgun sequencing that are old. We've spot checked the gDNA on tapestation and many samples have no visable DNA, many of the rest are sheared (DIN >5). We tried standard protocol using picogreen quants of 200ng and had no successful libraries. Tech support suggests treating these samples as if they were FFPE and sent a paper suggesting increased PCR cycles and increased bead ratio in the cleanup.

She also suggested reducing tagmentation time, but won't give any guidance on how much. Anyone have experience with playing with tagmentation time? how much do you reduce it? The protocol says 15 min. Drop to 10min? 7.5min?
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
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