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iGEM Bioware. Summer Week 7!. Overview. Arsenic Gold Decoder Lock & Key Other. Arsenic Collection System. Most PCRs didn't work (10% success rate!), and we're waiting on primers and restriction enzymes. The PCRs that worked showed ArsR and pArsR: - PowerPoint PPT Presentation

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  • iGEM BiowareSummer Week 7!

  • OverviewArsenicGoldDecoderLock & KeyOther

  • Arsenic Collection SystemMost PCRs didn't work(10% successrate!), and we're waiting on primers and restriction enzymes. The PCRs that worked showed ArsR and pArsR:

    Also we digested Amp backbone so we are ready to ligate in ArsR.Transformed pArsR into pSB1T3 this AM.1kb ArsR!1kb pArsR 100bp with primers F-255, R-23kb2kb1.5kb1kb

    .5kb

  • Gel of pSB1T3 and pArsR digestions

    We did a gel extract, ligation,and then transformed cells this morning.Undigested/Single Digest

    Vector Backbone

    Insert (RFP)pArsR3kb2kb1.5kb1kb

    .5kb

  • Gold Collection SystemGolB - digested, ligated into pSBIA2GolS - digested, ligated into pSBIA2GesABC - ?GolT - ?

  • Recent attempt to clone out GesABC and GolT withGolB and GolS as positive controlsThe gel on right shows bands only in lanes with no template.GolB-TGolT-TGolS-T

  • Gold Collection SystemStill trying to clone out GesABC and GolTRan PCR program with elongated extension time (12 min) 1. Denature @ 92 degC for 2 min 2. Denature @ 92 degC for 30 sec 3. Annealing @ 51 degC for 30 sec 4. Extension @ 68 degC for 12 min 5. Repeat from step 2 for 30 cycles 6. Final Extension @ 68 degC for 10 min

    GesABC was a 50 ul reaction 2 ul PrimerF, 2 ul PrimerR, 2 ul polymerase, 2 ul dNTP, 16 ul template, 5 ul buffer, 21 ul H2O

  • (7.8.10 gel)

  • Digestion, Gel extraction*full digest with XbaI and SpeI unless otherwise indicated

    *blue boxes show which bands we cut out

  • Yesterday, we ran gradient PCR with different annealing temperatures

    Tried 48, 50, and 52 degC for GesABC and GolT 51, 53, and 55 degC for GolB and GolS

  • DecodersRNA target biobricks have put into pSB1AC3F (RFC25).OmpFOmpAGadXNew Primers have been orderedto fit sRNA target biobricks into pSB1A3 (RFC 10).

    sRNA biobricks have been put into pSB1T3MicFMicAGadYThese regulators will have 8 extra nucleotides in their 5' end (biobrick scar)

    When will we have the computer back for the plate reader?

  • Lock and Key DecoderSpent the majority of this week waiting on restriction enzymes.

  • QuestionsPlate reader?

    Is there a way to prevent the gel from being half dark?

    Nano-drop and PCR purification consistency?

    Other Updates

    Haven't heard back from Midsci, but Brian Gallagher from GE Healthcare called about donating products.

    Volleyball tournament postponed until just after school starts.