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iGEM Bioware

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:

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iGEM Bioware

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  1. iGEM Bioware Summer Week 7!

  2. Overview Arsenic Gold Decoder Lock & Key Other

  3. 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: Also we digested Amp backbone so we are ready to ligate in ArsR. Transformed pArsR into pSB1T3 this AM. 1kb                pArsR          100bp                 with primers                   F-255, R-2 1kb   ArsR! 3kb 2kb 1.5kb 1kb .5kb

  4. 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) 3kb 2kb 1.5kb 1kb .5kb pArsR

  5. Gold Collection System GolB - digested, ligated into pSBIA2 GolS - digested, ligated into pSBIA2 GesABC - ? GolT - ?

  6. Recent attempt to clone out GesABC and GolT with GolB and GolS as positive controls The gel on right shows bands only in lanes with no template. GolT -T GolB -T GolS -T

  7. Gold Collection System Still trying to clone out GesABC and GolT Ran 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

  8.  (7.8.10 gel)

  9. Digestion, Gel extraction *full digest with XbaI and SpeI unless otherwise indicated *blue boxes show which bands we cut out

  10. 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

  11. Decoder sRNA target biobricks have put into pSB1AC3F (RFC25). OmpF OmpA GadX New Primers have been ordered to fit sRNA target biobricks into pSB1A3 (RFC 10).   sRNA biobricks have been put into pSB1T3 MicF MicA GadY These regulators will have 8 extra nucleotides in their 5' end (biobrick scar) When will we have the computer back for the plate reader?

  12. Lock and Key Decoder    Spent the majority of this week waiting on restriction enzymes.  

  13. Questions Plate 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.

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