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Two emulsion PCRs??

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  • Two emulsion PCRs??

    Hi!!

    New to the 454.

    I need to know this: for shotgun sequencing, if I want to do a bulk run: do you do 2 emulsion PCRs??? One with the SV kit, to do the titration (up until % enrichment bead calculation) and another 6hr amplification for the bulk run (with the library quantities obtained from the titration enrichment?

    THANKS A LOT!

  • #2
    Originally posted by dmrodz View Post
    Hi!!

    New to the 454.

    I need to know this: for shotgun sequencing, if I want to do a bulk run: do you do 2 emulsion PCRs??? One with the SV kit, to do the titration (up until % enrichment bead calculation) and another 6hr amplification for the bulk run (with the library quantities obtained from the titration enrichment?

    THANKS A LOT!
    That is certainly the safest way to go. In our experience, starting at 0.5 to 1 cpb works most but not all the time and it's sad to waste a large volume emPCR kit and get 35% enrichment. We do 8 SV reactions at 4 input amounts.

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    • #3
      Thanks a lot

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