Team:Nevada/Week 3

From 2012.igem.org

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==June 4==
==June 4==
 +
:Jasmine:
 +
:::Created primer stocks for J23116 Promoter, Terminator, RFP sense, RFP antisense
 +
:::Used Phusion PCR to create expression plasmid (EP) and RFP gene
 +
 +
:Michelle and Joe:
 +
:::PCR colony check of transformation SBP (SpeI and PstI) and LRP (PstI and XbaI)
 +
:::Cultured two colonies that expressed the brightest bands on the gel with Terrific Broth-Kanamyacin (TB-KAN) overnight.
 +
 +
:Jeremiah & Chris:
 +
:::Prepared new primers for PCR.
 +
:::Colony PCR of SBP-TBP
 +
:::::Antisense: TBP-anti534 , Sense: SBP-sense124
 +
 +
:Justin and Dafne:
 +
:::Ligate B12 into SBP.   
==June 5==
==June 5==
 +
:Michelle and Joe:
 +
:::Miniprep and prepared cultures colonies #1 and #2 from transformation SBP (SpeI and PstI)and LRP (PstI and XbaI).
 +
 +
:Jeremiah & Chris:
 +
:::Purification of TSAP-SBP
 +
:::PCR of SBP-TBP, then culture in stocks #3 & #4 TB broth for twenty-three hours.
==June 6==
==June 6==
 +
:Jasmine:
 +
:::PCR Purified EP and RFP
 +
:::Digested EP with DpnI
 +
:::Self-ligated EP
 +
 +
:Michelle and Joe:
 +
:::Digest colonies #1 and #2 from transformation SBP (SpeI and PstI) and LRP (PstI and XbaI)with enzymes SpeI and XbaI.
 +
 +
:Jeremiah & Chris:
 +
:::Digested sample #4 for use in expression plasmids with RFP with XbaI and SpeI
 +
:::Cultured samples #1 & #2, PCR of #2
 +
:::::Antisense: TBP-anti534 , Sense: SBP-sense124
 +
:::::Sample #2 sent to NV genomic center for sequencing
 +
 +
:Justin and Dafne:
 +
:::Transform SBP-B12 ligation into TOP10 E. coli cells
==June 7==
==June 7==
 +
:Jasmine:
 +
:::Transformed ligated EP into competent cells
 +
:::Plated cells onto Amp plate
 +
 +
:Michelle and Joe:
 +
:::Digest SBP + LRP with SpeI and PstI.
 +
:::Digest Red Fluorescent Protein (RFP) with XbaI and PstI and also with EcoRI and PstI.
 +
 
 +
:Jeremiah & Chris:
 +
:::Digestion of SBP-TBP – will be combined with VB12 binding protein with SpeI & PstI
 +
:::Cultured more of sample #4 SBP-TBP – will be used for all future stock
 +
 +
:Justin and Dafne:
 +
:::Colony  check-PCR of SBP-B12 colonies
 +
:::Culture successful colonies in TB-kana
==June 8==
==June 8==
 +
:Michelle and Joe:
 +
:::Check digests: SBP + LRP with SpeI and PstI, RFP with XbaI and PstI, and RFP backbone with EcoRI and PstI.
 +
:::PCR purification of RFP (XbaI and PstI).
 +
:::Digest SBP + LRP with EcoRI and PstI.
 +
 
 +
:Jeremiah & Chris:
 +
:::Digestion with EcoRI and PstI of SBP-TBP to be ligated with RFP   
 +
:::Digestion of VB12 with XbaI and PstI to be ligated into SBP-TBP
 +
       
 +
:Justin and Dafne:
 +
:::Miniprep cultures, run on 1.2% agarose gel to confirm successful plasmid transformation
 +
:::Once confirmed, digest SBP-B12 plasmid with Xba I and Pst I HF
 +
:::Digest overnight

Latest revision as of 01:25, 27 October 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 | Final Weeks |


Week 3: June 4 - June 8

Contents

June 4

Jasmine:
Created primer stocks for J23116 Promoter, Terminator, RFP sense, RFP antisense
Used Phusion PCR to create expression plasmid (EP) and RFP gene
Michelle and Joe:
PCR colony check of transformation SBP (SpeI and PstI) and LRP (PstI and XbaI)
Cultured two colonies that expressed the brightest bands on the gel with Terrific Broth-Kanamyacin (TB-KAN) overnight.
Jeremiah & Chris:
Prepared new primers for PCR.
Colony PCR of SBP-TBP
Antisense: TBP-anti534 , Sense: SBP-sense124
Justin and Dafne:
Ligate B12 into SBP.

June 5

Michelle and Joe:
Miniprep and prepared cultures colonies #1 and #2 from transformation SBP (SpeI and PstI)and LRP (PstI and XbaI).
Jeremiah & Chris:
Purification of TSAP-SBP
PCR of SBP-TBP, then culture in stocks #3 & #4 TB broth for twenty-three hours.

June 6

Jasmine:
PCR Purified EP and RFP
Digested EP with DpnI
Self-ligated EP
Michelle and Joe:
Digest colonies #1 and #2 from transformation SBP (SpeI and PstI) and LRP (PstI and XbaI)with enzymes SpeI and XbaI.
Jeremiah & Chris:
Digested sample #4 for use in expression plasmids with RFP with XbaI and SpeI
Cultured samples #1 & #2, PCR of #2
Antisense: TBP-anti534 , Sense: SBP-sense124
Sample #2 sent to NV genomic center for sequencing
Justin and Dafne:
Transform SBP-B12 ligation into TOP10 E. coli cells

June 7

Jasmine:
Transformed ligated EP into competent cells
Plated cells onto Amp plate
Michelle and Joe:
Digest SBP + LRP with SpeI and PstI.
Digest Red Fluorescent Protein (RFP) with XbaI and PstI and also with EcoRI and PstI.
Jeremiah & Chris:
Digestion of SBP-TBP – will be combined with VB12 binding protein with SpeI & PstI
Cultured more of sample #4 SBP-TBP – will be used for all future stock
Justin and Dafne:
Colony check-PCR of SBP-B12 colonies
Culture successful colonies in TB-kana

June 8

Michelle and Joe:
Check digests: SBP + LRP with SpeI and PstI, RFP with XbaI and PstI, and RFP backbone with EcoRI and PstI.
PCR purification of RFP (XbaI and PstI).
Digest SBP + LRP with EcoRI and PstI.
Jeremiah & Chris:
Digestion with EcoRI and PstI of SBP-TBP to be ligated with RFP
Digestion of VB12 with XbaI and PstI to be ligated into SBP-TBP
Justin and Dafne:
Miniprep cultures, run on 1.2% agarose gel to confirm successful plasmid transformation
Once confirmed, digest SBP-B12 plasmid with Xba I and Pst I HF
Digest overnight

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