Team:Nevada/Week 8
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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/ | + | [[Team:Nevada/Final Weeks| Final Weeks]] | |
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==July 9== | ==July 9== | ||
- | + | :Jasmine & Joe | |
+ | :::Checked 10 CP-EP* colonies with colony PCR | ||
+ | :::Cultured colonies 5 and 7 in TB-Amp | ||
+ | :Michelle | ||
+ | :::Culture SBP-LRP plasmid | ||
+ | :Jeremiah & Chris: | ||
+ | :::TBP+++ purified and digested (gel 066) | ||
+ | :::Digestion of SBP-TBP to use with new expression vector | ||
+ | ::::XbaI and PstI | ||
+ | :Justin & Dafne | ||
+ | :::Ligate SBP into SBP-B12 plasmid | ||
+ | :::Transform into TOP 10 competent cells | ||
==July 10== | ==July 10== | ||
+ | :Jasmine and Joe: | ||
+ | :::Miniprepped CP-EP* cultures | ||
+ | :::Digested CP-EP* with SpeI and NsiI | ||
+ | :::Dephosphorylated CP-EP* digestion | ||
+ | :::Ligated RFP* with CP-EP* | ||
+ | :::Created new primer stocks for VR, VF2, and a new EP*-anti-SpeI | ||
+ | :::Amplified new EP (EP^), lac promoter, and tet promoter using PCR | ||
+ | |||
+ | :Michelle: | ||
+ | :::Grew SBP-LRP plasmid from transformation of SBP (SpeI and PstI) and LRP (PstI and XbaI) | ||
+ | :::Miniprep of SBP-LRP plasmid from transformation of SBP (SpeI and PstI) and LRP (PstI and XbaI), nanodrop, and :::pellet since 1st miniprep concentration was too little. Pellet was then miniprepped and nanodropped. | ||
+ | :::Digest transformation SBP (SpeI and PstI) + LRP (PstI and XbaI) with XBA I and PST I, | ||
+ | :::run through gel, and ligate with EP. | ||
+ | |||
+ | :Jeremiah & Chris: | ||
+ | :::Ligation of SBP-TBP à ExV using new method | ||
+ | |||
+ | :Justin and Dafne | ||
+ | :::SBP-B12-SBP transformation successful | ||
+ | :::Multiple colonies grew, and colony PCR check confirms | ||
+ | :::SBP-B12 in expression plasmid turned out to be unsuccessful | ||
+ | :::Digest SBP-B12 plasmid by Xba I and Pst I HF | ||
+ | :::Ligate into new expression plasmid | ||
+ | |||
==July 11== | ==July 11== | ||
+ | :Jasmine and Joe: | ||
+ | :::Transformed ligated RFP*-CP-EP* into competent cells and plated onto 1 amp plate with L-arabinose and one without | ||
+ | :::Transformed original CP plasmid into competent cells and plated onto 1 amp plate with L-arabinose and one without | ||
+ | :::Digested lac promoter and tet promoter with EcoRI and SpeI | ||
+ | |||
+ | :Michelle: | ||
+ | :::Transformation of ligation SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid digested with | ||
+ | :::XBA I and PST I and EP onto AMP plates. | ||
+ | |||
+ | :Jeremiah & Chris: | ||
+ | :::Transformed SBP-TBP-ExV | ||
+ | :::Colonies were too numerous to count (TNTC) | ||
+ | |||
+ | :Justin and Dafne | ||
+ | :::Transform ligation of SBP-B12 insert into expression | ||
+ | :::TOP10 competent cells were used | ||
+ | |||
==July 12== | ==July 12== | ||
+ | :Michelle: | ||
+ | :::Check transformation of SBP (Spe1 and PstI)-LRP (PstI and XbaI) digested with XbaI and PstI + EP on AMP plates. | ||
+ | :::PCR colony check 24 colonies with Forward primer (Control promoter) and Reverse primer (Terminator) and ran gel. | ||
+ | :Jeremiah & Chris: | ||
+ | :::Colony PCR of plate #1 | ||
+ | :::No positive colonies or failed PCR | ||
+ | |||
+ | :Justin and Dafne | ||
+ | :::Colony PCR check of colonies produced by SBP-B12 in expression plasmid transformation | ||
+ | :::Colony check successful | ||
+ | :::Culture successful colony in LB-amp overnight | ||
==July 13== | ==July 13== | ||
+ | :Michelle: | ||
+ | :::PCR check colony #9 from transformation of SBP (SpeI and PstI)-LRP (PstI and | ||
+ | :::XbaI) digested with XbaI and PstI + EP with Forward primer (Control promoter) and Reverse | ||
+ | :::primer (Lysine antisense) and ran gel to double check if PCR colony #9 is truly successful. | ||
+ | :::Cultured 2 new colonies from 7/10 KAN plates containing successful transformation of SBP (SpeI and PstI) and LRP :::PstI and XbaI) with TB-KAN broth. | ||
+ | |||
+ | :Jeremiah & Chris: | ||
+ | :::Colony PCR of both plates again | ||
+ | :::Culturing colony 1-9 from plate #1 and colony 2-4 from plate #2 | ||
+ | :::Ligation of SBP-TBP into constitutive vector – transformed on 07/15 | ||
+ | |||
+ | :Justin and Dafne | ||
+ | :::once OD of the over night culture reached 0.6, L-arabinose was added in varying concetrations | ||
+ | ::::Tube 1: 0.001% Tube 2: 0.01% Tube 3: 0.1% Tube 4: 1.0% | ||
+ | :::Control: SBP-B12 expression plasmid not induced by L-arabinose | ||
+ | :::After 4 hours, time sample 1 was pelleted and placed in -80 freezer | ||
+ | :::After 8 hours, time sample 2 was pelleted and placed in -80 freezer |
Latest revision as of 01:35, 27 October 2012
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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 9
- Jasmine & Joe
- Checked 10 CP-EP* colonies with colony PCR
- Cultured colonies 5 and 7 in TB-Amp
- Michelle
- Culture SBP-LRP plasmid
- Jeremiah & Chris:
- TBP+++ purified and digested (gel 066)
- Digestion of SBP-TBP to use with new expression vector
- XbaI and PstI
- Justin & Dafne
- Ligate SBP into SBP-B12 plasmid
- Transform into TOP 10 competent cells
July 10
- Jasmine and Joe:
- Miniprepped CP-EP* cultures
- Digested CP-EP* with SpeI and NsiI
- Dephosphorylated CP-EP* digestion
- Ligated RFP* with CP-EP*
- Created new primer stocks for VR, VF2, and a new EP*-anti-SpeI
- Amplified new EP (EP^), lac promoter, and tet promoter using PCR
- Michelle:
- Grew SBP-LRP plasmid from transformation of SBP (SpeI and PstI) and LRP (PstI and XbaI)
- Miniprep of SBP-LRP plasmid from transformation of SBP (SpeI and PstI) and LRP (PstI and XbaI), nanodrop, and :::pellet since 1st miniprep concentration was too little. Pellet was then miniprepped and nanodropped.
- Digest transformation SBP (SpeI and PstI) + LRP (PstI and XbaI) with XBA I and PST I,
- run through gel, and ligate with EP.
- Jeremiah & Chris:
- Ligation of SBP-TBP à ExV using new method
- Justin and Dafne
- SBP-B12-SBP transformation successful
- Multiple colonies grew, and colony PCR check confirms
- SBP-B12 in expression plasmid turned out to be unsuccessful
- Digest SBP-B12 plasmid by Xba I and Pst I HF
- Ligate into new expression plasmid
July 11
- Jasmine and Joe:
- Transformed ligated RFP*-CP-EP* into competent cells and plated onto 1 amp plate with L-arabinose and one without
- Transformed original CP plasmid into competent cells and plated onto 1 amp plate with L-arabinose and one without
- Digested lac promoter and tet promoter with EcoRI and SpeI
- Michelle:
- Transformation of ligation SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid digested with
- XBA I and PST I and EP onto AMP plates.
- Jeremiah & Chris:
- Transformed SBP-TBP-ExV
- Colonies were too numerous to count (TNTC)
- Justin and Dafne
- Transform ligation of SBP-B12 insert into expression
- TOP10 competent cells were used
July 12
- Michelle:
- Check transformation of SBP (Spe1 and PstI)-LRP (PstI and XbaI) digested with XbaI and PstI + EP on AMP plates.
- PCR colony check 24 colonies with Forward primer (Control promoter) and Reverse primer (Terminator) and ran gel.
- Jeremiah & Chris:
- Colony PCR of plate #1
- No positive colonies or failed PCR
- Justin and Dafne
- Colony PCR check of colonies produced by SBP-B12 in expression plasmid transformation
- Colony check successful
- Culture successful colony in LB-amp overnight
July 13
- Michelle:
- PCR check colony #9 from transformation of SBP (SpeI and PstI)-LRP (PstI and
- XbaI) digested with XbaI and PstI + EP with Forward primer (Control promoter) and Reverse
- primer (Lysine antisense) and ran gel to double check if PCR colony #9 is truly successful.
- Cultured 2 new colonies from 7/10 KAN plates containing successful transformation of SBP (SpeI and PstI) and LRP :::PstI and XbaI) with TB-KAN broth.
- Jeremiah & Chris:
- Colony PCR of both plates again
- Culturing colony 1-9 from plate #1 and colony 2-4 from plate #2
- Ligation of SBP-TBP into constitutive vector – transformed on 07/15
- Justin and Dafne
- once OD of the over night culture reached 0.6, L-arabinose was added in varying concetrations
- Tube 1: 0.001% Tube 2: 0.01% Tube 3: 0.1% Tube 4: 1.0%
- Control: SBP-B12 expression plasmid not induced by L-arabinose
- After 4 hours, time sample 1 was pelleted and placed in -80 freezer
- After 8 hours, time sample 2 was pelleted and placed in -80 freezer
- once OD of the over night culture reached 0.6, L-arabinose was added in varying concetrations