Team:Nevada/Week 14

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==August 21==
==August 21==
 +
:Michelle:
 +
 +
:::Western blot transfer of remaining supernatant from last Western blot transfer of small colony from transformation :::of M2-BL21.
 +
 +
:Justin and Dafne:
 +
:::Obtain protein from cells as done before
 +
:::Purify protein using Ni-NTX nickel column (per protocol)
 +
:::Analyze using western blot and coomassie brilliant blue
 +
 +
:Jeremiah & Chris:
 +
:::Western blot development (BL21 cells with no thiamine added)
 +
:::No band appeared after development so IPTG promoter could be the problem (picture on iphone)
 +
:::Cancel other expression attempt using IPTG and added thiamine
 +
:::Attempt to express our gene by use of a TET promoter
 +
:::Set up phusion PCR for the TBP+ gene
==August 22==
==August 22==
 +
:Joe:
 +
:::Colony PCR - gel 171 right side both colonies good
 +
 +
:Michelle:
 +
:::Western blot transfer continued from yesterday for remaining supernatant sample for small colony from transformation :::of M2-BL21. Membrane developed.
 +
:::Cultured more M2-BL21 with LB AMP-CM.
 +
 +
:Jeremiah & Chris:
 +
:::Ran gel of phusion PCR products to confirm existence (gel #169)
 +
:::Purify gene using glass milk protocol
 +
:::Ran gel to see concentration (gel #170)
 +
:::Ligate phusion products together
 +
:::Expression plasmid phusion product already don
==August 23==
==August 23==
 +
:Joe:
 +
:::miniprep of SBP-RFP cultures
 +
:::PCR - gel 173 SBP-RFP-Tet[f] bad, Tet[f] backbone good
 +
:::culture more RFP plasmid
 +
 +
:Michelle:
 +
:::Glycerol stock 1 ml of M2-BL21 + LB AMP-CM.
 +
:::Protein purification: Pellet 40 ml of M2-BL21 + LB AMP-CM, sonic pulse, follow procedure
 +
:::for Nickel (Ni) column to purify protein.
 +
 +
:Jeremiah & Chris:
 +
:::Trans formation failed due to use of the wrong cell line (BL21 cells instead of TOP 10)
 +
:::Attempt transformation again
==August 24==
==August 24==
 +
:Joe:   
 +
:::miniprep RFP
 +
:::new primers: RFPEPftanti50, RFPEPftsense50, RFPEPresense60, RFPEPreanti60
 +
::::SBPftsense, SBPftanti, SBPresense, SBPreanti
 +
:::PCR sample of each - gel 174 show all but RFPft good
 +
:::PCR purify
 +
:::DPN digest of RFP
 +
 +
:Michelle:
 +
:::Coomassie staining to detect purified protein, SBP-LRP.
 +
 +
:Jeremiah & Chris:
 +
:::Transformation failed again
 +
:::Purified fresh TET backbone phusion product
 +
:::Ran on a gel (gel #176) to confirm concentration
 +
:::Glass milk purify TBP+ phusion product gene
 +
 +
==August 25==
 +
:Michelle:
 +
:::SDS-PAGE Coomassie staining of gel. 
 +
:::Large-scale expression of protein purification: Cultured 1 ml of M2-BL21 glycerol stock
 +
:::into 500 ml of LB AMP-CM.
 +
 +
:Jeremiah & Chris:
 +
:::Ligated phusion products together (TET and TBP+)
 +
:::Transformed into TOP 10 cells

Latest revision as of 01:38, 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 14: August 20 - August 25

Contents

August 20

Joe:
gel 167 - RFP pcr good, gel purify
Digestions: SBP(SpeI/PstI), RFP(XbaI/PstI)
Ligation of two digestions
Michelle:
Western blot transfer continued from 8/17 for samples of small colony from transformation of M2-BL21 developed :::membrane.
Justin and Dafne:
Large scale expression (200ml) of L-arabinose induced SBP-B12 protein with 5 nM vitamin B-complex
Jeremiah & Chris:
Western blot protocol (BL21 cells with no thiamine added)
Run time points on a polyacrylamide gel (BL21 cells with thiamine added)

August 21

Michelle:
Western blot transfer of remaining supernatant from last Western blot transfer of small colony from transformation :::of M2-BL21.
Justin and Dafne:
Obtain protein from cells as done before
Purify protein using Ni-NTX nickel column (per protocol)
Analyze using western blot and coomassie brilliant blue
Jeremiah & Chris:
Western blot development (BL21 cells with no thiamine added)
No band appeared after development so IPTG promoter could be the problem (picture on iphone)
Cancel other expression attempt using IPTG and added thiamine
Attempt to express our gene by use of a TET promoter
Set up phusion PCR for the TBP+ gene

August 22

Joe:
Colony PCR - gel 171 right side both colonies good
Michelle:
Western blot transfer continued from yesterday for remaining supernatant sample for small colony from transformation :::of M2-BL21. Membrane developed.
Cultured more M2-BL21 with LB AMP-CM.
Jeremiah & Chris:
Ran gel of phusion PCR products to confirm existence (gel #169)
Purify gene using glass milk protocol
Ran gel to see concentration (gel #170)
Ligate phusion products together
Expression plasmid phusion product already don

August 23

Joe:
miniprep of SBP-RFP cultures
PCR - gel 173 SBP-RFP-Tet[f] bad, Tet[f] backbone good
culture more RFP plasmid
Michelle:
Glycerol stock 1 ml of M2-BL21 + LB AMP-CM.
Protein purification: Pellet 40 ml of M2-BL21 + LB AMP-CM, sonic pulse, follow procedure
for Nickel (Ni) column to purify protein.
Jeremiah & Chris:
Trans formation failed due to use of the wrong cell line (BL21 cells instead of TOP 10)
Attempt transformation again

August 24

Joe:
miniprep RFP
new primers: RFPEPftanti50, RFPEPftsense50, RFPEPresense60, RFPEPreanti60
SBPftsense, SBPftanti, SBPresense, SBPreanti
PCR sample of each - gel 174 show all but RFPft good
PCR purify
DPN digest of RFP
Michelle:
Coomassie staining to detect purified protein, SBP-LRP.
Jeremiah & Chris:
Transformation failed again
Purified fresh TET backbone phusion product
Ran on a gel (gel #176) to confirm concentration
Glass milk purify TBP+ phusion product gene

August 25

Michelle:
SDS-PAGE Coomassie staining of gel.
Large-scale expression of protein purification: Cultured 1 ml of M2-BL21 glycerol stock
into 500 ml of LB AMP-CM.
Jeremiah & Chris:
Ligated phusion products together (TET and TBP+)
Transformed into TOP 10 cells

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