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Team:Nevada/Week 9
From 2012.igem.org
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 | Final Weeks |
Contents |
July 16
- Michelle:
- Miniprep from yesterday’s cultures of SBP (SpeI and PstI)-LRP (PstI and XbaI) and digest with XbaI and PstI.
- Chris & Jermiah
- Colony PCR ligation from 07/15 (gel 089)
- Most likely switching to IPTG promoter
- Samples from colony 1-9 set up with L. Arabinose expression gradient.
- 1.0 mM, 0.1 mM, 0.01 mM, 0.001 mM
- Samples were taken in intervals of hours: 0,2,4,6,8, O/N
- Colony PCR ligation from 07/15 (gel 089)
- Justin and Dafne
- Overnight samples were taken for all concentrations
- Run samples using SDS-PAGE
- Placed gel #1 in coomassie Brilliant blue for 1.5 hours and destained using 30% methanol solution
- Performed Western Blot with gel #2
July 17
- Joe: Ligation Tet with EP*, IPTG with EP*
- Transform ligations
- digestion of RFP=EP^ by SpeI and Nsi
- sequence RFP*-EP^
- Michelle:
- Run gel of SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid digested with XbaI and PstI. Ligation of SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid digested with XbaI and PstI with EP onto AMP plate followed by transformation.
- Jeremiah & Chris:
- Load page gels with all samples
- Unable to run gel due to technical difficulties
- Load page gels with all samples
- Justin and Dafne
- Develop Western blot using TMB- Microwell Peroxidase
- Bands developed at 41 kDa for 0.1% overnight sample
- Next stage: medium scale expression
July 18
- Joe:RFP*EP^ miniprep
- ligation of RFP*EP^ with SBP
- Transform
- Colony PCR of IPTG-Ep^ and Tet-EP^ plates
- Michelle:
- PCR colony check 12 colonies from yesterday’s transformation of SBP (SpeI and PstI)-LRP
- (PstI and XbaI) plasmid digested with XbaI and PstI with EP on AMP plate using Forward
- primer (Constitutive promoter-J32119) and Reverse primer (Lysine antisense) and ran gel.
- Cultured colony #2 from transformation with SBP (SpeI and PstI)-LRP (PstI and XbaI)
- digested with XbaI and PstI ligated with EP in TB-AMP
- Jeremiah & Chris:
- Purified and sent samples to NV Genomic Center for sequencing
- TBP+++
- SBP-TBPàExV
- Purified and sent samples to NV Genomic Center for sequencing
- Justin and Dafne
- Cultured 200 ml of SBP-B12 bacteria
July 19
- Joe: Culture IPTG colonies 1, 4, 5, 6 and TET colonies 1, 2, 3, 4 PCS RFP*-EP^
- Michelle:
- Miniprep cultured colony #2 from transformation of SBP (SpeI and PstI)-LRP (PstI and XbaI)
- digested with XbaI and PstI ligated with EP from yesterday. Added Forward primer
- (Constitutive promoter-J32119) and Reverse Primer (Lysine antisense) for sequencing at Nevada Genomic Center.
- Digest colony #2 from transformation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested
- with XbaI and PstI ligated with EP with enzymes, EcoRI and PstI.
- Jeremiah & Chris:
- Ligated TBP into SBP on the other side of the SBP than done originally (+TBP)
- Justin and Dafne
- Spin down culture, resuspend in lysis buffer, and lyse cells with sonicator: 8 rounds of 10 seconds on and 10 :::seconds off
- Pellet
- seperate supernatant from pellet
- Analyze pellet and supernatant using SDS-PAGE followed by Western Blot
July 20
- Joe: PCR of RFP*-Ep^
- miniprep of IPTG and TET cultures
- sequencing of IPTG and TET samples
- NHE digestion of samples
- Michelle:
- Ran gel of yesterday’s digestion with EcoRI and PstI of colony #2 from transformation of SBP
- (SpeI and PstI)-LRP (PstI and XbaI) digested with XbaI and PstI ligated with EP.
- Western blot transfer of 2 ml of colony #2 pellet from transformation of SBP (SpeI and PstI)-LRP (PstI and XbaI) :::digested with XbaI and PstI ligated with EP.
