We were thinking of using rolling circle amplification of plasmids to generate the DNA we would use to make Nextera libraries for high plexity pools to load on a MiSeq. We have succeeded in sequencing 384 plasmids in one run and are moving to 1536 and beyond. Has anyone out there tried RCA with the Nextera process?
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Hi Creeves
I wondering why you are using RCA with Nextera. Nextera XT have been designed for library preparation directly from plasmids among other types of DNA. I see RCA a shortcut to avoid plasmid preps. -
The reason is simply cost reduction. We want base pair resolution QC of the thousands of constructs we routinely make in our automated strain engineering pipeline. The MiSeq can reliably sequence about 2000 10-20 kb plasmids in one run, but at that scale the plasmid prep cost becomes an issue.Comment
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