Team:Nevada/Week 8

From 2012.igem.org

(Difference between revisions)
Line 38: Line 38:
:::Checked 10 CP-EP* colonies with colony PCR
:::Checked 10 CP-EP* colonies with colony PCR
:::Cultured colonies 5 and 7 in TB-Amp
:::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 10==
 
==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

Revision as of 18:59, 25 September 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 | Week 20 | Week 21


Week 8: July 9 - July 13

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

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