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Old 12-16-2019, 10:50 AM   #1
drdna
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Location: Kentucky

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Default Expired AMPURE XP

Does anyone have any experience as to what happens when AMPURE XP reagent goes bad? We have been using it routinely to purify high molecular weight DNA for Nanopore sequencing but also of a sudden, we're getting zero DNA recovery. This is associated with expensive clumping of the beads after incubation with the DNA, and a failure of the bead clumps to dissociate during "elution."

Im looking for information on how "expired" beads normally behave: do they fail to bind DNA in the first place, or do they do what we're seeing: hold on to it so tightly that it's impossible to release. I don't need suggestions of things to try to release the DNA - we've tried EVERYTHING. Just needing to know how to tell if beads have gone bad - one of our bottles is only 8 months old.
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Old 12-18-2019, 04:55 PM   #2
teuthbrush
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thread: http://seqanswers.com/forums/showthread.php?t=8347

Not very familiar with Nanopore workflow but:

Is this in any way digested DNA? The BSA in some restriction enzymes makes it very difficult to resuspend the beads. I've found this is particularly true if you're working in 96 well format and using a low-volume magnetic plate -- it just makes this smear (rather than a ring) across the inside of the tube and will not resuspend no matter how much pipetting you do.

For what it's worth, I've worked with Ampure that is pretty expired (5-6 months past expiration) without problems.

I'd try using it on a ladder and seeing what turns up in terms of yield and size selection, in any case.
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Old 12-19-2019, 10:30 AM   #3
angelas
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Are you working with DNA from an unusual source or a different extraction protocol? I have had beads respond similarly (clumpy and then won't pellet) when working with kelp DNA that was contaminated with polysaccharides. I had to dilute the DNA and then let the beads stay on a magnet overnight to pellet.
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