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Team:Nevada/Week 3
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 |
June 4
- Jasmine:
- Created primer stocks for J23116 Promoter, Terminator, RFP sense, RFP antisense
- Used Phusion PCR to create expression plasmid (EP) and RFP gene
- Michelle and Joe:
- PCR colony check of transformation SBP (SpeI and PstI) and LRP (PstI and XbaI)
- Cultured two colonies that expressed the brightest bands on the gel with Terrific Broth-Kanamyacin (TB-KAN) overnight.
- Jeremiah & Chris:
- Prepared new primers for PCR.
- Colony PCR of SBP-TBP
- Antisense: TBP-anti534 , Sense: SBP-sense124
- Justin and Dafne:
- Ligate B12 into SBP.
June 5
- Michelle and Joe:
- Miniprep and prepared cultures colonies #1 and #2 from transformation SBP (SpeI and PstI)and LRP (PstI and XbaI).
- Jeremiah & Chris:
- Purification of TSAP-SBP
- PCR of SBP-TBP, then culture in stocks #3 & #4 TB broth for twenty-three hours.
June 6
- Jasmine:
- PCR Purified EP and RFP
- Digested EP with DpnI
- Self-ligated EP
- Michelle and Joe:
- Digest colonies #1 and #2 from transformation SBP (SpeI and PstI) and LRP (PstI and XbaI)with enzymes SpeI and XbaI.
- Jeremiah & Chris:
- Digested sample #4 for use in expression plasmids with RFP with XbaI and SpeI
- Cultured samples #1 & #2, PCR of #2
- Antisense: TBP-anti534 , Sense: SBP-sense124
- Sample #2 sent to NV genomic center for sequencing
- Justin and Dafne:
- Transform SBP-B12 ligation into TOP10 E. coli cells
June 7
- Jasmine:
- Transformed ligated EP into competent cells
- Plated cells onto Amp plate
- Michelle and Joe:
- Digest SBP + LRP with SpeI and PstI.
- Digest Red Fluorescent Protein (RFP) with XbaI and PstI and also with EcoRI and PstI.
- Jeremiah & Chris:
- Digestion of SBP-TBP – will be combined with VB12 binding protein with SpeI & PstI
- Cultured more of sample #4 SBP-TBP – will be used for all future stock
- Justin and Dafne:
- Colony check-PCR of SBP-B12 colonies
- Culture successful colonies in TB-kana
June 8
- Michelle and Joe:
- Check digests: SBP + LRP with SpeI and PstI, RFP with XbaI and PstI, and RFP backbone with EcoRI and PstI.
- PCR purification of RFP (XbaI and PstI).
- Digest SBP + LRP with EcoRI and PstI.
- Jeremiah & Chris:
- Digestion with EcoRI and PstI of SBP-TBP to be ligated with RFP
- Digestion of VB12 with XbaI and PstI to be ligated into SBP-TBP
- Justin and Dafne:
- Miniprep cultures, run on 1.2% agarose gel to confirm successful plasmid transformation
- Once confirmed, digest SBP-B12 plasmid with Xba I and Pst I HF
- Digest overnight
