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  • E120 reactions

    Hello All, is anyone running E120 reactions? I've tried 2 now and each time I only have 400M beads after enrichment instead of 2.2B.

    We are using the EZ bead system and following the aqueous phase set up as per the calculator provided by AB (with a total of around 4.4ng of template, ave size 290bp).

    What are the key factors in getting a successful reaction? if the instruments are set up correctly and the input material is correct what else can I tweak?

    JPC

  • #2
    E120

    Hi,

    I have done several successful E120s, but when I have a problem it is usually too many beads...

    How are you quantifying your library? If this is not accurate, then you will never get good beads.

    Are you using a scale to prepare your oil mix or using the 6 falcon tubes? I heard some labs were having trouble with scales, so that's the reason for the falcon tube method.

    Do you see any beads falling out of emulsion after amplification? Beads seen at bottom of pouch?

    Just a few things I can think of...

    Richard

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    • #3
      You probably should try qPCR on the library. EZ Bead tends to give some beads, even with no library (unlike the manual method). Your library may not be good.

      --
      Phillip

      Comment


      • #4
        Thanks for the replies, we are quantifying with the Qubit, diluting our sample down to around 35pg/ul so that we are adding 125 ul of a 290bp product, I didn't want to dilute any further in case we drop below the qubit HS range.

        'Yes' we are prep'ing in batches of 6 falcon tubes and 'No' we don't see nay bead fall out at all.

        I'll give the qPCR a go to check the library, I have no reason to think there is a problem with it (we check regularly with the bioabnalyser, nanodrop and qubit) but it seems like a worthwhile exercise.

        thanks both

        JC

        Comment


        • #5
          Originally posted by JPC View Post
          Hello All, is anyone running E120 reactions? I've tried 2 now and each time I only have 400M beads after enrichment instead of 2.2B.

          We are using the EZ bead system and following the aqueous phase set up as per the calculator provided by AB (with a total of around 4.4ng of template, ave size 290bp).

          What are the key factors in getting a successful reaction? if the instruments are set up correctly and the input material is correct what else can I tweak?

          JPC
          I have heard recently from my FAS that one of the improvements to the enricher with the EZBead upgrade to v1.9, which has just been released, is to filter the E120 scale beads in several batches. Currently it filters in one batch and with the large scale emulsions there are just too many beads which puts excessive pressure on the filter causing it to be inefficient.
          If you feel your libraries are OK then maybe this is your problem and you are loosing 'good' beads.

          Yvonne

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          • #6
            We were told by AB to increase our titration point to 0.6picomoles this means our input library amount went from 3.62ng to 4.85ng for a 239 bp library. Unfortunately the Enricher (now running v1.9) shook the Hyb tube from it's place holder and the resulting Output tube solution is cloudy and not very brown....waiting to here from AB if I can collect the beads back together and save the emPCR somehow?!

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