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Primer dimer in ChIP-seq library

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  • Primer dimer in ChIP-seq library

    I saw primer dimer in my library on bioanalyzer. If I do not want to re make the library, should I re do the size selection or purify it with ampure beads? Which one is better (cleaner and recovers more)? Thanks a lot!

  • #2
    Purify with Ampure XP at 1:1 - 1.2:1. We use 1:1 based on the Illumina mRNAseq prep recommendations, another contributor on the site (ETHANol) has made an absolutely excellent CHIP-seq protocol available and used 1.2:1 as a final purification step.

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    • #3
      Thanks a million!

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      • #4
        Which is 1.2x, the AmPure or the sample?

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        • #5
          Normally AMPure and then sample.

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          • #6
            That is correct, for absolute clarification;

            1 volume Ampure XP : 1 volume Library

            for an equally good separation of the dimer with a bit better recovery, no idea of this is true but I've come to trust ETHANol's suggestions

            1.2 volumes Ampure XP : 1 volume library

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            • #7
              I have one question about this, if you purify directly the library, do you have enough complexity? because you are amplifying many primer molecules instead DNA molecules.

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              • #8
                The complexity of your library would be limited by the number of amplifiable library molecules pre-amplification. If you happen to be able to see the library at this point on a bioanalyzer high sensitivity chip, you could estimate your maximum complexity. 100 pg is about 100 million 1 kbp molecules.

                One the one hand, it would be better to remove the adapter dimer before PCR, so you don't get any of it annealing to full length library molecules and escaping later size selection.

                On the other hand, if you do the PCR first, then the amount of DNA you are working with is higher. Generally it is easier to deal with higher concentration DNA than lower concentration. (At least in the low nM range and below.)

                --
                Phillip

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