Team:Nevada/Week 11

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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 |


Week 11: July 30 - August 3

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