Team:Nevada/Week 15

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