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  • Illumina PE library prep with AmpureXP

    Has anyone been successful at making a standard Illumina PE library using AmpureXP beads for clean up and size selection (no columns & no gels)? If so, was size selection carried out before PCR or after? I have been trying this, and my samples look fine until post amplification. Samples are not amplifying! My agilent traces of prePCR samples, look fine. There are no large adaptor peaks that lead me to believe that the adaptors have not ligated properly. I have tried size selection and multiple ethanol washes before PCR, but to no avail. Any suggestions?

  • #2
    Sadly your pre-PCR agilent trace would only tell you that the fragmentation worked. Any or all of the steps after fragmentation could have failed: blunting, adenylation, ligation -- even your PCR may have been the problem. Generally a reaction step can fail because of contaminants (eg. ethanol or denaturants) introduced during post reaction "clean-up".

    I have not made one of these libraries, but the protocol mentions that "a molar excess of adapter to fragments is used", so you might expect to see adapter peaks.

    --
    Phillip

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    • #3
      We've used the Pippin prep for size selection then cleaned up with AMPure at the end and had great success. Maybe you can fine tune your size selection step?

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      • #4
        We regularly do all of our cleanups and size selections by Ampure XP beads. The only tricky part is account for the PEG in your ligation buffer if you are going straight into size selection.

        Pretty much the only time we do gels anymore is when we want a very tight fragment distribution.

        I would suggest optimizing it in your lab with your buffers, etc. It is great once you have it working. Oh, and there is slight variance between lots of beads, so you may want to re-validate each lot.

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        • #5
          I am not having trouble getting my library in the correct size range. Rather, it just isn't amplifying. After I have size selected, I run an agilent chip and I see a nice peak, but after amplification my trace is a flat line.

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          • #6
            Yeah, to me that says your ligation failed for some reason. Failure to blunt, failure to a-tail or failure of the ligation itself. Either contamination of the sample with substances that inhibit one of those reactions or some component missing from one of those reactions.

            On the other hand, it seems like you should be able to see at least the original template you added to the PCR amplification. What type of agilent chip are you using?

            Can you run a bit of your PCR reaction on an agarose gel -- just to make sure your BioAnalyzer isn't on the fritz?
            --
            Phillip

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