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Team:Nevada/Week 6
From 2012.igem.org
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[[Team:Nevada/Week 18| Week 18]] | | [[Team:Nevada/Week 18| Week 18]] | | ||
[[Team:Nevada/Week 19| Week 19]] | | [[Team:Nevada/Week 19| Week 19]] | | ||
| - | + | [[Team:Nevada/Final Weeks| Final Weeks]] | | |
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Revision as of 01:26, 27 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 | Final Weeks |
Contents |
June 25
- Jasmine:
- Cultured more EP in TB-Amp
- Michelle and Joe:
- Re-run PCR colony samples of RFP-TA colony #2 (XbaI and PstI) transformation from 6/22.
- Cultured colonies #7, 8, 9 from PCR colony samples RFP-TA colony #2 (XbaI and PstI) with TB-AMP.
- Transformation of ligation of SBP + LRP (XbaI, EcoRI, SpeI, and PstI) with EP from 6/22 on X-gal + AMP plate.
- Jeremiah & Chris:
- Transformation of SBP-TBP-VB12
- Justin and Dafne:
- At this point, the unsuccessful nature of our project lead to the restart from the point of ligating SBP-B12 insert into the expression plasmid
- New Miniprep buffers were used
- New ligase was used
June 26
- Jasmine:
- Miniprepped new EP cultures
- Digested EP with SpeI
- Michelle and Joe:
- Check transformation of SBP + LRP (XbaI, EcoRI, SpeI, and PstI) with EP on X-gal + AMP plate.
June 27
- Jasmine, Joe, and Michelle:
- Run EP digest on agarose gel to check
- Created primer stocks for new RFP (RFP*), new EP (EP*), and new controlled promoter (CP): RFP*-sense-SPEI-NsiI, RFP*-anti-PstI, EP*-sense-NsiI, EP*-anti-SpeI, CP-sense-EcoRI, CP-anti-SpeI
- Amplified EP* using Phusion PCR
- Blunted EP* with Blunting Enzyme
- Jeremiah & Chris:
- Colony PCR of transformation from 6/25 (gel 040) – good yield on all lanes
- Cultured samples 1 & 8
- Phusion PCR of SBP-TBP in expression vector
- Justin and Dafne:
- SBP “fusion” into SBP-B12 plasmid.
- SBP-B12 was digested using Xba I and Pst I HF
- PCR was used to add 15 bp to the end of both SBP and the open SBP-B12 plasmid
- This process allowed for the annealing of SBP and SBP-B12 to occur without the normal ligation process
- SBP-B12 insert was also ligated into the new expression plasmid using this technique
June 28
- Jasmine, Joe, and Michelle:
- Self-ligated blunted EP*
- Transformed EP* into competent cells and plated onto Amp plate
- Jeremiah & Chris:
- Purified samples of 1 and 8 of SBP-TBP-VB12
- 1 – 194ng/ul #2 – 239ng/ul
- Samples 1 and 8 sent to NV Genomic Center for Sequencing
- Purified LRP already digested (XbaI and PstI)
- SBP-TBP-VB12 (TBP++) digested to ligate with LRP
- Ligation of LRP à TBP++
- Purified samples of 1 and 8 of SBP-TBP-VB12
- Justin Emlen:
- Transform the SBP-B12 insert in the expression plasmid into TOP 10 competent cells
June 29
- Jasmine, Joe, and Michelle:
- Checked 8 EP* colonies with colony PCR
- Cultured colonies 2 and 6 in TB-Amp
- Amplified RFP* and CP using PCR
- Digested RFP* with SpeI, PstI, and DpnI
- Jeremiah & Chris:
- Transformation of LRP-TBP++
- Phusion transformation of SBP-TBP à expression plasmid on LB-Amp
- Justin and Dafne:
- Colony PCR check SBP-B12 insert in expression plasmid
- Three successful colonies were obtained
- Culture colonies in 6 ml of TB-amp
