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Team:Nevada/Week 4
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
Contents |
June 11
- Jeremiah & Chris:
- Purified and digested samples of both #2 and #4 with XbaI and SpeI
- Ran on gel to ensure existence of desired plasmid
- Digestions on SBP, TBP, RFP, and SBP-TBP failed.
- Ligated RFP à SBP
- Ligated VB12 à SBP-TBP
- SBP-TBP à expression vector
- Jasmine:
- Checked EP plate colonies using colony PCR
- Grew cultures of colony #5 and #7
- Michelle and Joe:
- Check digestions: SBP + LRP (EcoRI and PstI) and PCR clean up/check of RFP (XbaI and PstI).
- Ligate 6/6 expression digest: SBP (SpeI and PstI) and LRP (PstI and XbaI) digested with SpeI and XbaI with RFP backbone.
- Digest RFP with XbaI and PstI.
- Dephosphorylate SBP + LRP (SpeI and PstI).
- Justin and Dafne:
- Ligate SBP-B12 digest into Expression plasmid
- Expression plasmid was digested and dephosphorylated by Jasmine
- Ligation took place overnight
June 12
- Michelle and Joe:
- Redigest RFP with XbaI and PstI.
- Transformation of ligation of 6/6 expression digest: SBP (SpeI and PstI) and LRP (PstI and XbaI) digested with SpeI and XbaI with RFP backbone into competent cells onto Ampicillin (AMP) plates.
- Jeremiah & Chris:
- Transformations of ligations from 6/11
- SBP-TBP-VB12 --> LB + Kan
- SBP-TBP-ExVector --> LB + Amp
- SBP-RFP --> LB +Ka
- Repeat of ligations from 6/11 – incubated overnight.
- Transformations of ligations from 6/11
- Justin and Dafne:
- Digest SBP-B12 plasmid by Xba I and Spe I
- For the creation of SBP-B12-SBP Plasmid
- This project was not seen to its entirety
- Digest SBP-B12 plasmid by Xba I and Spe I
June 13
- Jasmine:
- Miniprepped EP cultures
- Digested EP with SpeI
- Digested RFP with XbaI and SpeI
- Dephosphorylated digested EP
- Ligated EP and RFP
- Michelle and Joe:
- Check transformation of 6/6 expression digest: SBP (SpeI and PstI) and LRP (PstI and XbaI) digested with SpeI and XbaI with RFP backbone into competent cells onto Ampicillin (AMP) plates.
- Digest SBP + LRP with four enzymes: XbaI, EcoRI, PstI, and SpeI and PCR followed by PCR purification and PCR.
- Jeremiah & Chris:
- Transformations of ligations from 6/12
- SBP-TBP-VB12 on LB + Ka
- SBP-TBP-ExVector on LB + Amp
- SBP-RFP on LB +Ka
- Digested SBP-TBP again for use with expression vector with XbaI, SpeI, PstI, EcoRI
- Transformations of ligations from 6/12
- Justin and Dafne:
- Ligate SBP-B12 insert (Xba I and Spe) into Expression plasmid
June 14
- Jasmine:
- Transformed RFP-EP ligation into competent cells
- Plated transformation onto Amp plates
- Michelle and Joe:
- Ligation of digestion SBP + LRP (XbaI, EcoRI, PstI, and SpeI) from yesterday with RFP backbone using ligation protocol and Roche method.
- PCR and PCR purify RFP before ligating using Invitrogen TA ligation kit followed by transformation.
- Jeremiah & Chris:
- PCR cleaned ligations from 6/13
- Transformed SBP-TBP-ExV àLB+Amp (only one colony grew)
- Digested SBP+TBP and ligated with expression vector using both standard and Roche methods. (Having problems getting SBP-TBP to express, hypothesis is that proteins in too high concentration are fatal to cells)
- Justin and Dafne:
- Transform expression plasmid with SBP-B12 insert
- This transformation did not give positive results
June 15
- Jasmine:
- Checked RFP-EP plate
- Michelle and Joe:
- Check transformation of RFP ligation from yesterday. PCR colony check colonies from RFP
- TA transformation plate and ran through gel to check colonies.
- Tranformation of ligation SBP + LRP (XbaI, EcoRI, PstI, and SpeI) with RFP backbone onto AMP plates.
- Jeremiah & Chris:
- Colony PCR of single colony grown.
- Transformation of Roche and standard ligations from 6/14 onto LB-Amp
- Justin and Dafne:
- Digest SBP-B12 plasmid with Xba I and Pst I HF
- Ligate into new expression plasmid created by Jasmine
