Team:Nevada/Week 17

September 10

Michelle:

Single colony isolation of M2-BL21 from LB AMP-CM stock plate, which was followed by culturing it in 10ml LB :::AMP-CM broth and then large scale culturing with 50ml of LB AMP-CM broth.

Justin and Dafne:

Proteins were separated from cells (as done before)

Suspended in Amylose column buffer

proteins were purified using Amylose Resin High Flow (NEB)

eluted using 1M glycine: pH 10 buffer

September 11

Joe:

harvest protein from RFP-SBP

Justin and Dafne

Concentrate protein using Desalting column

Jeremiah & Chris:

Express BL21 cells by use of a L. Arabanose gradient

Took and 8 hour and overnight time sample

September 12

· Run samples on a polyacrylamide gel · Western blot protocol

September 13

Joe: Add rice to purified protein, incubate for 5 hours

   Not enough protein - did not work

Michelle:

   Glycerol stocked M2-BL21 before large scale culturing M2-BL21 with 50ml LB AMP-CM broth.

September 14

Joe: make 10, 10 ml cultures of RFP-SBP in Top10 cells

Michelle:

   Transformed M2 (SBP-LRP) into BL21 cells and plated on LB AMP-CM plates.

Jeremiah & Chris:

        Develop western
       No band appeared because the BL21 cells needed to be grown in Ampicillin (Amp) and Chloramphenicol (Cm) antibiotics
       Minipreped fresh expression plasmid with TBP+ from BL21 culture
       Ran on a gel (gel #193)
       Transform plasmid into new BL21 cells
       Use Amp and Cm antibiotics when plating
       Prepare submission plasmid (pBP31C)
       Miniprep and digest in large quantities as to have enough for the other groups

Chris and Jeremiah:

     Colony PCR check 5 samples