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John Tyson 2020. Modified LSK109 ligation prep with needle shear and bead clean up. protocols.io https://dx.doi.org/10.17504/protocols.io.7emhjc6Copy Citation Copied
URL: https://dx.doi.org/DOI:10.17504/protocols.io.7emhjc6
Authors: John Tyson
Summary: Modified LSK109 ligation prep using needle shear and bead purificationUsing HMW DNA purified as above we have been performing 29G and 26G needle shears to produce fragmentation to around 18Kb and 34kb respectively (reported read N50’s after basecalling). Also by going into the library preparations with 5-10ug of DNA have been able to produce enough material to provide up to 10 library loads from a single prep. We have not been performing the FFPE repair during the end-prep step, and have been diluting the sample at the cleanup points in the protocol with EB to prevent bead clumping during 0.4-0.5x AMPureXP purifications when DNA fragments are large and at high concentrations. Shown below are the results from a batch of 12 runs we performed using this approach.
Affiliations: Snutch Lab, UBC, Vancouver, BC, Canada
External URL: https://www.longreadclub.org/mountain-protocol/
Version: 1
Publication Date: 2020
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