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  • pmiguel
    replied
    Did you see no reduction in the amount of 100bp band compared to the 300bp band with a 0.9X AMPure? There should have been some, although it will not have been a complete removal of the band.

    --
    Phillip

    Leave a comment:


  • ojm101
    replied
    Thanks very much pmiguel.

    When I mentioned the homebrew system, I was really referring to the incoming reaction compositions. These tests were all with AMPure beads.

    Reducing the ratio makes sense but my target fragment size is ~200 bp before ligation and 300 bp after ligation. As such, I worry about losses with a ratio as low as 0.5x.

    I have seen many gel images posted on this website and elsewhere that showed distinct losses of bands of ladder with reducing the bead ratio. The fact that there is any trouble reproducing what should be very straightforward is troubling.

    Does anybody have any literature or advice on HOW the factors pmiguel listed (temperature, time, etc) can affect binding? Could any of them account for what I am seeing, which appears to be binding all the way down to 100 bp regardless of ratio?

    Leave a comment:


  • pmiguel
    replied
    We calibrate our batches of AMPure and generally see a 50-80% decrease in the amount of a 100 bp band with 0.9X volumes of AMPure. We always do a series of calibrations down to 0.5X volumes. You should probably do the same for your home brew.

    Keep in mind it isn't the beads themselves that effect the size cut, but the PEG concentration of the solution and other factors that effect PEG precipitations (eg, temperature, time). So if you are using "home brew" beads it could be your PEG is a little more concentrated than you intended.

    --
    Phillip

    Leave a comment:


  • ojm101
    started a topic AMPure adapter dimer carryover THROUGH CAPTURE

    AMPure adapter dimer carryover THROUGH CAPTURE

    Hi All,

    I have been having some trouble with 120 bp adapter dimers showing up after ligation during Illumina library prep. We have a homebrew system here, and previously we incorporated SPRIselect (distinct from AMPure XP) but we are phasing that out because of the low yield issues and subsequent bias. As soon as we took out SPRIselect, we started seeing adapter dimers all over the place, which prompted us to use a lower (<1.8x) ratio of AMPure beads to "cut" the dimers out.

    And that is where the confusion started. I used 100 bp ladder and 0.9x and 1.2x ratios of ampure beads and basically got no cutoff whatsoever. I could still see the 100 bp band in all samples. I am confident I used the correct volumes of beads and that there was no evaporation/discrepancy regarding my ladder samples. The ladder was in 50 uL and the beads were 45 and 60 uL respectively.

    What else could be going on here? It's occurred to me that the lot of these beads might have some slight variation but not such that I would still be seeing 100 bp fragments with a 0.9x ratio. I have seen a lot of people using reduced binding ratios and getting results that I have thus far been unable to reproduce. Based on these results it's no wonder that adapter dimers are making it through in my libraries.

    What's even crazier is that these dimers are somehow finding their way into our probe captured libraries! Has this happened to anyone before?

    Let's please assume that all pipetting was done accurately and there is no adapter contamination anywhere in the lab. We have considered this and taken every measure possible to avoid it and I do not want this thread to focus on contamination problems unless you feel like you have something extremely profound to share.

    Thanks in advance

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