Click here to load reader
Post on 13-Jan-2016
Embed Size (px)
DESCRIPTIONiGEM 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
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
Gel of pSB1T3 and pArsR digestions
We did a gel extract, ligation,and then transformed cells this morning.Undigested/Single Digest
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
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.
Is there a way to prevent the gel from being half dark?
Nano-drop and PCR purification consistency?
Haven't heard back from Midsci, but Brian Gallagher from GE Healthcare called about donating products.
Volleyball tournament postponed until just after school starts.