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Old 04-02-2019, 05:38 PM   #1
cement_head
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Default Bead removal of <1000 bp from LARGE fragments

Hi,

Trying to prep some very large fragment libraries (>50 kbp), and I've isolated the gDNA using a BluePippin, but when I use beads (Promega ProNex) to clean up, or rather, remove the small fragments (<1000 bp), I get low yield of the input 50 kbp gDNA.

Are there specific beads that are better for this? AMPure XP PB? Suggestions?

Thanks,
Andor
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Old 04-02-2019, 08:42 PM   #2
luc
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The beads are likely all very similar. You could try to elute for a much longer time than usual for short fragments.
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Old 04-03-2019, 05:18 AM   #3
cement_head
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No, I don't want the small fragments. I want only fragments LARGER than 1000 bp.

In essence, I want to eliminate any small (unseen) fragments that may unduly influence a nanopore/ONT minION library prep.

Thanks
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Old 04-04-2019, 03:25 AM   #4
nucacidhunter
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Loss of large fragments mostly can be due to inefficient binding. longer incubation of bead-DNA solution on a rotating wheel should increase recovery.
Quantifying supernatant with PicoGreen or Qubit dsDNA HS assay can point if this is the case.

Circulomics short read eliminator kit is also a good choice.
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Old 04-04-2019, 12:00 PM   #5
luc
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You misunderstood me: Long fragments will require a longer time to elute/dissolve from the beads compared to shorter fragments.

Quote:
Originally Posted by cement_head View Post
No, I don't want the small fragments. I want only fragments LARGER than 1000 bp.

In essence, I want to eliminate any small (unseen) fragments that may unduly influence a nanopore/ONT minION library prep.

Thanks
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ampure beads, large inserts, pronex, remove fragments

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