Team:Nevada/Week 6

From 2012.igem.org

(Difference between revisions)
Line 35: Line 35:
==June 25==
==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==
==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==
==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==
==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++
 +
 +
:Justin Emlen:
 +
:::Transform the SBP-B12 insert in the expression plasmid into TOP 10 competent cells
==June 29==
==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

Revision as of 22:10, 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 6: June 25 - June 29

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. 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++
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

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