Team:Nevada/Week 4

From 2012.igem.org

(Difference between revisions)
Line 35: Line 35:
==June 11==
==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==
==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.
 +
 +
: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
==June 13==
==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
 +
 +
:Justin and Dafne:
 +
:::Ligate SBP-B12 insert (Xba I and Spe) into Expression plasmid
==June 14==
==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==
==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

Revision as of 21:52, 22 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 4: June 11 - June 15

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.
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

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
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

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