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Old 08-28-2012, 06:36 AM   #1
crocky
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Location: Switzerland

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Default Alternative DNA elution for ChIPseq

Dear all,
I am performing ChIPseq experiments, and I am looking for an alternative for phenol-chloroform-isoamylalcohol elution/precipitation after the IP. I have tried Qiagen columns, but the recovery is 1/10 of phenol:chloroform:isoamylalcohol (measured by Qubit) . As I am quite restricted in starting material I cannot use more . Does anyone of you have experience with AMPURE XP beads at this step? I am wondering whether they will preform well, as DNA concentration is rather low (should be ~0.05 ng/Ál at this step), and size is rather low as well (50 to >1000 bp, but enrichment between 100 - 500 bp).

Reasons that I want to omit the phenol stuff is problems with automated subsequent library preparation caused by tiniest amounts of residual phenol, and that I am pregnant.

Thanks a lot for your help!
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Old 08-30-2012, 05:37 AM   #2
Veleno
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Diagenode IPure works well for me; the only downside is that you recover more background crap so you may want to be a bit more stringent in your washes.
It's cheaper than the Qiagen per reaction too and does not perform any size selection.
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Old 08-30-2012, 06:44 AM   #3
Chipper
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Ampure works with low conc (I have sucessfully used it for library preps where it was below the Qubit detection limit) but I am not sure if recovery is affected by the SDS concentration in the elution buffer. I would try with a lower elution volume (eg 50 ul), and perhaps test the recovery with some input DNA at varying concentration in IPEB first.
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Old 10-15-2012, 01:15 AM   #4
epigeneticfan
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I use Diagenode IPure after ChIP and MeDIP. My concentration is usually 1 ng per Ál after MeDIP but i managed to purify a fragment of 350 bp at the concentration of 2.7 ng in 100 Ál with a recovery of 75% compare to phenol-chloroform. Moreover, the elution buffer of IPure kit doesn’t contain any detergent and salt which is useful for any downstream application like library preparation. Based on my own experience, I did not observe any increased background when comparing IPure to Qiagen column purification for the H3K4me3 histone mark.
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