Team:Nevada/Week 15

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Week 15: August 27 - August 31

Contents

August 27

Joe: fusion and transformation
Michelle:
Pellet large scale expression to prepare for purification of M2-BL21 glycerol stock
+ LB AMP-CM.

LJustin and Dafne:

Determine optimal Imidazole concentration to utilize in Ni-column purification
Create 12 mM, 14 mM, 16 mM Imidazole binding buffers
Three seperate protein expressions were carried out (as done above)
Each pellet was suspended in one of the binding buffers and then the cells were lysed using the sonicator (as :::done before)
The supernatant of each different binding buffer was run through the Ni-column
Jeremiah & Chris:
Transformation failed
Set up phusion for TET plasmid
Ligate the phusion products (TET and TBP+)

August 28

Joe:
Redigestion of RFPEP-R by DpnI
PCR of SBP-R, RFP-R using longamp
Michelle:
Purification of large scale expression of M2-BL21 glycerol stock + LB AMP-CM using a Nickel column.
Conducted SDS-PAGE Coomassie and Western Transfer Blot protocol, which continued until tomorrow.
Justin and Dafne:
Western blot analysis of above protein purification
Western blot analysis was uninspiring, indicating the concentration change did not effect purification
Jeremiah & Chris:
Run ligation on a gel (gel #176)
Gel showed no ligation occurred
Phusion PCR the TET plasmid and TBP+ gene (Long Amp Taq)
Ligate the phusion products
Transform into TOP 10 cells

August 29

Michelle:
SDS-PAGE Coomassie staining of gel followed by desalting of protein at ~30kDa, which was successful.

August 30

Joe:
PCR clean up - gel 173 good
Transform, plate on AMP
Michelle:
Develop Western Transfer Blot of purified large scale expression of M2-BL21 glycerol stock + LB AMP-CM.
Modified ELISA assay using starch and testing with iodine to check the presence of starch in wells. Purified :::SBP-LRP also tested with Starch-ELISA.

August 31

Joe:
Colony PCR of RFPEP-R