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Alternative DNA elution for ChIPseq

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  • 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/µl 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!

  • #2
    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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    • #3
      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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      • #4
        I use Diagenode IPure after ChIP and MeDIP. My concentration is usually 1 ng per µl after MeDIP but i managed to purify a fragment of 350 bp at the concentration of 2.7 ng in 100 µl 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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