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Old 09-28-2020, 12:49 AM   #1
Junior Member
Location: Zurich

Join Date: Sep 2020
Posts: 2
Default AMPure XP clumps during Cas9 library prep

Hi everyone,
I'm using the ONT CRISPR-Cas9 targeted enrichment protocol ( to sequence a 18kb locus in DNA which I extracted from human brain using the Circulomics Nanobind kit (which is recommended for this application). The protocol has worked well on a few occasions (around 120x coverage, which I understand is all right for this application), but on other occasions the coverage was quite poor (e.g. 6x), despite the DNA being very high quality (>60kb on Genomic Tapestation).

I suspect that the low coverage stems from a problem during the library prep:

After Cas9-directed cleavage of DNA and ligation of ONT adapters, the library is purified with 0.3x AMPure XP beads. I have observed on several occasions that the beads form insoluble clumps the moment that they are added to the tube, and I suspect that much of the library is lost within the clump. I have tried prolonged incubation and gentle agitation, but I couldn't dissolve the clump, neither in the ONT Long Frament Buffer, nor in the elution buffer.

Has someone got any tips on how to avoid this issue?
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Old 01-18-2022, 06:32 PM   #2
David Eccles (gringer)
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 843

Try needle-shearing your DNA prior to doing the CRISPR-Cas9 digest, e.g. 20 passes with a 26G or 29G needle (see here for more information). This has been working well for us to make the samples easier to handle and quantify.
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ampure, cas9, crispr, nanopore

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