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Team:Nevada/Week 11
From 2012.igem.org
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Revision as of 03:54, 4 October 2012
Protocols | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 |
Contents |
July 30
- Joe:
- Transform SBP-RFP to EP-CP
- Plate on AMP
- TET/IPTG- miniprep of cultures
- Dafne and Michelle:
- Digest CP-EP* L-arabinose with SpeI and NSI).
- Digest SBP (SpeI and PstI)-LRP (PstI and XbaI) with SpeI and EcoRI.
- PCR purification of L-arabinose EP and ran gel.
- Justin and Dafne
- Removed dialysis membrane from 8M urea solution, placed in 6M urea solution
- Run another expression to determine if there is a concentration of L-arabinose that could be used in order to :::induce protein expression without causing it to be moved to inclusion body
- SDS-PAGE and Western blot analysis confirmed that SBP-B12 protein will be moved to inclusion body no matter the :::L-arabinose concentration
July 31
- Joe:
- SBP-RFP in EP-CP plates show no growth, PCR more
- PCR purification - gel 120 bad
- PCR more Ep-Cp
- PCR purification - gel 121 compared before and after purification - Purification bad
- PCR more Ep-Cp - gel 123 bad
- Digestion ep-cp by Nsi/SpeI
- Michelle:
- Miniprep, PCR using sense primer (DNA2PTFsense) and antisense primer (DNA2PTFanti),
- PCR purification, run sample with PCR purification and one without PCR purification through
- gel, and digest SBP (SpeI and PstI)-LRP (PstI and XbaI) that was PCR purified with XbaI and PstI.
- Cultured 2 colonies of CP-EP* with L-arabinose with TB-AMP.
- Justin and Dafne
- Place dialysis membrane in 4M Urea buffer
August 1
- Joe:
- Ep-Cp PCR, purify by glass fiber purification
- Gel 125 shows good purification
- Primer stocks- terminatior, tetrbs, lacrbs
- PCR of each part - gel 125 good
- Digest each by Nsi/SpeI
- Michelle:
- Run gel of yesterday’s digestion SBP (SpeI and PstI)-LRP (PstI and XbaI) with XbaI and PstI
- and dephosphorylate.
- Miniprep CP-EP* with L-arabinose, nanodrop, and digest with SpeI and NSI.
- PCR SBP (SpeI and PstI)-LRP (PstI and XbaI) in preparation for PCR purification tomorrow
- using glass milk.
- Justin and Dafne:
- Place dialysis membrane in 2M urea buffer
August 2
- Joe:
- Gel 126 shows all digestions successful
- 4cut digestion (EcoRI, XbaI, SpeI, PstI) of SBP-RFP
- Ligations- 4cut with tetrbs/terminatior, 4cut with lacrbs/terminator,
- Ep-Cp with SBP-RFP(XbaI/PstI)
- Michelle:
- PCR purification of SBP (SpeI and PstI)-LRP (PstI and XbaI) using glass milk.
- Run gel of CP-EP* L-arabinose (SpeI and NSI) and dephosphorylate.
- Ligate digestion of SBP (SpeI and PstI)-LRP (PstI and XbaI) with XbaI and PstI with CP-EP*
- L-arabinose (SpeI and NSI.
August 3
- Joe:
- Transform ligations
- PCR of ligations - gel 128 wrong sizes
- Digestion of SBP-RFP by SpeI/PstI
- Ligation of SBP-RFP(SpeI/PstI) to terminator
- Michelle:
- Transformation of ligation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested with XbaI and
- PstI and CP-EP* L-arabinose (SpeI and NSI) onto AMP plate.
- Justin and Dafne
- Place dialysis membrane in pure water
