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Problems with emulsion. Anyone have protocol changes?

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  • Problems with emulsion. Anyone have protocol changes?

    Hello,

    My lab is currently having trouble getting 70-80% template positive ISP for our Post-Enrichment product.(Our data is MUCH lower)

    It is almost a random occurrence when some emulsions will provide excellent results and some are very complete failures.

    We are wondering if anyone can attribute these hicupps to certain steps in the protocol or if anyone has found a particular reason for this anomaly. I would like to find a way to test our samples before running them in the sequencer to determine if our run will be a success or a failure.

    Any ideas or thoughts are welcome

    -MDnextgenseq

  • #2
    Which is it?: Your enrichment level is too high if you are able to enrich 70-80% of the total beads, then either there is too much library going into the emPCR or your emulsions are breaking, OR is it that the beads you are enriching are not 70-80% template positive.

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    • #3
      The beads we are enriching are not 70-80% they are much lower. Ie: our cy5 is extremely low on occasion. Our Fam is also low, leading me to believe that we are losing the majority of our beads somewhere.

      Not quite sure how to troubleshoot this yet, we have had some success and some failures using the same protocol.

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      • #4
        Welcome the fun world of trouble shooting emPCR! Are you able to count the number of beads going in and the number of beads being recovered? The failure could come from an incorrect number of beads going in, emulsions failing (so less DNA driven to beads), problems with the enrichment, etc. The options are almost endless... Was their tech support much help?

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        • #5
          Someone else on our team spoke with them and they suggested we still run the ion torrent dispite the bad emPCR and Bead Recovery.
          Both library's ran ok....90K final reads approx so much better that we were expecting, but the 1.2M wells and using 280M ISP I would hope for better results.
          Hopefully OneTouch will make a big difference. I'll post back when the we do our next run in a couple weeks.

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          • #6
            That is what we have seen, lucky to geto 10% of the wells give good signal. It makes you think about the number of beads needed for the 318 chip and how many reads it will actually get. They may need to be 500+ bp to hit 1Gb.

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            • #7
              The insert size should be <150bp, otherwise the live ISPs rate is very low (we tried). Besides a proper library and ISPs ratio is key. The accuracy of quantification and sample type can both affect a proper titration of this ratio.

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              • #8
                We have trouble quantitating library and the ISPs. Do you use qPCR to measure library concentration before emPCR?

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                • #9
                  Originally posted by Hiro.Protagonist View Post
                  We have trouble quantitating library and the ISPs. Do you use qPCR to measure library concentration before emPCR?
                  Agilent 2100 Bioanalyzer

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                  • #10
                    Has anyone had problems with their reagents over time????? We might be finding a degradation of the reagents as they are frozen and thawed many times that is impeding the empcr

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                    • #11
                      Originally posted by MDnextgenseq View Post
                      Has anyone had problems with their reagents over time????? We might be finding a degradation of the reagents as they are frozen and thawed many times that is impeding the empcr
                      I don't have a PGM, but I can tell you that what you are describing is endemic to any reagent. A good standard practice is to aliquot the amounts you need for a typical experiment into separate tubes. Then store them all in the freezer/fridge as appropriate. That way, you only have one freeze/thaw cycle.

                      Don't forget to invert your tubes to mix them thoroughly prior to aliquoting or use.

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