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

Chris and Jeremiah:

· Colony PCR check 5 samples