Team:Nevada/Week 5

From 2012.igem.org

(Difference between revisions)
Line 35: Line 35:
==June 18==
==June 18==
 +
:Jeremiah & Chris:
 +
:::Ran gel of colony PCR sample (001)
 +
:::Cultured both standard and Roche colonies. (later found that colonies didn’t contain desired plasmids)
 +
:::Cultured VB12 from stock plasmid in gel in LB+Amp
 +
:::Purified VB12
 +
:::Digested SBP-TBP and VB12 to ligate together again.
 +
:::::SBP-TBP: SpeI & PstI
 +
:::::VB12: XbaI & PstI
 +
:::Ligated VB12àSBP-TBP
 +
 +
:Jasmine:
 +
:::Created control transformations and plated onto Amp plates:
 +
:::::Expression plasmid alone
 +
:::::EP digested with SpeI
 +
:::::Self-ligated EP digest
 +
:::::Dephosphorylated EP digest
 +
 +
:Michelle and Joe:
 +
:::PCR purification of RFP plasmid and run through gel.
 +
:::PCR purified RFP plasmid again because sterile water was not used.
 +
:::After PCR purification of RFP, ligation using Invitrogen TA ligation kit and transformation into competent cells onto AMP plates was carried out.
==June 19==
==June 19==
 +
:Jasmine:
 +
:::Checked control transformations
 +
 +
:Michelle and Joe:
 +
:::Check transformation of RFP TA AMP plate.
 +
:::PCR colony check 9 colonies from RFP TA transformation and ran through a gel.
 +
:::Cultured colony #2 from RFP TA transformation using Terrific broth-Ampicillin (TB-AMP).
 +
:::Cultured more SBP + LRP from 6/4 colony #2 from successful transformation SBP (SpeI and PstI) and LRP (PstI and XbaI) with TB-KAN for stock.
 +
:::Transformation of 6/4 colony #2 from transformation SBP (SpeI and PstI) and LRP (PstI and XbaI) into competent cells followed by transformation onto KAN plates for stock.
 +
 +
:Jeremiah & Chris:
 +
:::Transformed SBP-TBP-VB12
 +
:::Digested SBP-TBP to be used in future ligations with XbaI & SpeI
==June 20==
==June 20==
 +
:Jasmine:
 +
:::Amplified more RFP using Phusion PCR
 +
 +
:Michelle and Joe:
 +
:::Miniprep cultured colony #2 from RFP-TA and digested with XbaI and PstI.
 +
:::Ran RFP-TA colony #2 digested with XbaI and PstI, followed by PCR purification, PCR
 +
:::Check with gel, and ligation using Invitrogen TA ligation kit.
 +
:::Check transformation of colony #2 from SBP (SpeI and PstI) and LRP (PstI and XbaI).
 +
:::Cultured colonies from transformation of colony #2 from SBP (SpeI and PstI) and LRP (PstI and XbaI) with TB-KAN for stock.
 +
 
 +
:Jeremiah & Chris:
 +
:::Retransformed SBP-TBP-VB12 as first transformations didn’t take.
 +
:::Ligated SBP-TBP into expression vector.
==June 21==
==June 21==
 +
:Michelle and Joe:
 +
:::Transformation of ligation of RFP-TA colony #2 (XbaI and PstI) digest from yesterday into competent cells and onto X-GAL plates.
 +
:::Pellet and miniprep SBP + LRP. Digest SBP + LRP with XbaI, EcoRI, SpeI, and PstI for expression.
 +
:::PCR purify 20ul of digest and elute with 50ul ddH20, then run through gel.
 +
:::Redigest SBP + LRP with XbaI, EcoRI, SpeI, and PstI again.
 +
 +
:Jeremiah & Chris:
 +
:::Digestion of SBP-TBP to try ligation again with VB12
 +
:::Ligated SBP-TBP into expression vector (TSAP)
 +
 +
:Justin and Dafne:
 +
:::Ligate SBP-B12 insert into expression plasmid
 +
:::Transform ligation into TOP 10 competent cells
 +
:::Results still negative
==June 22==
==June 22==
 +
:Michelle and Joe:
 +
:::Check transformation of RFP-TA colony #2 (XbaI and PstI) followed by PCR colony check.
 +
:::Run redigested SBP + LRP (XbaI, EcoRI, SpeI, and PstI) through gel. PCR purification using 40ul of digest and elute twice with 15ul ddH20. Ligate to expression plasmid (EP).
 +
 +
:Jeremiah & Chris:
 +
:::Transformation of SBP-TBP à ExV
 +
:::Ligation of VB12 à SBP-TBP
 +
:::Colony PCR of transformation from 6/15 (gel 027)

Revision as of 22:02, 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 5: June 18 - June 22

Contents

June 18

Jeremiah & Chris:
Ran gel of colony PCR sample (001)
Cultured both standard and Roche colonies. (later found that colonies didn’t contain desired plasmids)
Cultured VB12 from stock plasmid in gel in LB+Amp
Purified VB12
Digested SBP-TBP and VB12 to ligate together again.
SBP-TBP: SpeI & PstI
VB12: XbaI & PstI
Ligated VB12àSBP-TBP
Jasmine:
Created control transformations and plated onto Amp plates:
Expression plasmid alone
EP digested with SpeI
Self-ligated EP digest
Dephosphorylated EP digest
Michelle and Joe:
PCR purification of RFP plasmid and run through gel.
PCR purified RFP plasmid again because sterile water was not used.
After PCR purification of RFP, ligation using Invitrogen TA ligation kit and transformation into competent cells onto AMP plates was carried out.

June 19

Jasmine:
Checked control transformations
Michelle and Joe:
Check transformation of RFP TA AMP plate.
PCR colony check 9 colonies from RFP TA transformation and ran through a gel.
Cultured colony #2 from RFP TA transformation using Terrific broth-Ampicillin (TB-AMP).
Cultured more SBP + LRP from 6/4 colony #2 from successful transformation SBP (SpeI and PstI) and LRP (PstI and XbaI) with TB-KAN for stock.
Transformation of 6/4 colony #2 from transformation SBP (SpeI and PstI) and LRP (PstI and XbaI) into competent cells followed by transformation onto KAN plates for stock.
Jeremiah & Chris:
Transformed SBP-TBP-VB12
Digested SBP-TBP to be used in future ligations with XbaI & SpeI

June 20

Jasmine:
Amplified more RFP using Phusion PCR
Michelle and Joe:
Miniprep cultured colony #2 from RFP-TA and digested with XbaI and PstI.
Ran RFP-TA colony #2 digested with XbaI and PstI, followed by PCR purification, PCR
Check with gel, and ligation using Invitrogen TA ligation kit.
Check transformation of colony #2 from SBP (SpeI and PstI) and LRP (PstI and XbaI).
Cultured colonies from transformation of colony #2 from SBP (SpeI and PstI) and LRP (PstI and XbaI) with TB-KAN for stock.
Jeremiah & Chris:
Retransformed SBP-TBP-VB12 as first transformations didn’t take.
Ligated SBP-TBP into expression vector.

June 21

Michelle and Joe:
Transformation of ligation of RFP-TA colony #2 (XbaI and PstI) digest from yesterday into competent cells and onto X-GAL plates.
Pellet and miniprep SBP + LRP. Digest SBP + LRP with XbaI, EcoRI, SpeI, and PstI for expression.
PCR purify 20ul of digest and elute with 50ul ddH20, then run through gel.
Redigest SBP + LRP with XbaI, EcoRI, SpeI, and PstI again.
Jeremiah & Chris:
Digestion of SBP-TBP to try ligation again with VB12
Ligated SBP-TBP into expression vector (TSAP)
Justin and Dafne:
Ligate SBP-B12 insert into expression plasmid
Transform ligation into TOP 10 competent cells
Results still negative

June 22

Michelle and Joe:
Check transformation of RFP-TA colony #2 (XbaI and PstI) followed by PCR colony check.
Run redigested SBP + LRP (XbaI, EcoRI, SpeI, and PstI) through gel. PCR purification using 40ul of digest and elute twice with 15ul ddH20. Ligate to expression plasmid (EP).
Jeremiah & Chris:
Transformation of SBP-TBP à ExV
Ligation of VB12 à SBP-TBP
Colony PCR of transformation from 6/15 (gel 027)

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