Team:Nevada/Week 8

July 9

Jasmine & Joe

Checked 10 CP-EP* colonies with colony PCR

Cultured colonies 5 and 7 in TB-Amp

Michelle

Culture SBP-LRP plasmid

Jeremiah & Chris:

TBP+++ purified and digested (gel 066)

Digestion of SBP-TBP to use with new expression vector

XbaI and PstI

Justin & Dafne

Ligate SBP into SBP-B12 plasmid

Transform into TOP 10 competent cells

July 10

Jasmine and Joe:

Miniprepped CP-EP* cultures

Digested CP-EP* with SpeI and NsiI

Dephosphorylated CP-EP* digestion

Ligated RFP* with CP-EP*

Created new primer stocks for VR, VF2, and a new EP*-anti-SpeI

Amplified new EP (EP^), lac promoter, and tet promoter using PCR

Michelle:

Grew SBP-LRP plasmid from transformation of SBP (SpeI and PstI) and LRP (PstI and XbaI)

Miniprep of SBP-LRP plasmid from transformation of SBP (SpeI and PstI) and LRP (PstI and XbaI), nanodrop, and :::pellet since 1st miniprep concentration was too little. Pellet was then miniprepped and nanodropped.

Digest transformation SBP (SpeI and PstI) + LRP (PstI and XbaI) with XBA I and PST I,

run through gel, and ligate with EP.

Jeremiah & Chris:

Ligation of SBP-TBP à ExV using new method

Justin and Dafne

SBP-B12-SBP transformation successful

Multiple colonies grew, and colony PCR check confirms

SBP-B12 in expression plasmid turned out to be unsuccessful

Digest SBP-B12 plasmid by Xba I and Pst I HF

Ligate into new expression plasmid

July 11

Jasmine and Joe:

Transformed ligated RFP*-CP-EP* into competent cells and plated onto 1 amp plate with L-arabinose and one without

Transformed original CP plasmid into competent cells and plated onto 1 amp plate with L-arabinose and one without

Digested lac promoter and tet promoter with EcoRI and SpeI

Michelle:

Transformation of ligation SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid digested with

XBA I and PST I and EP onto AMP plates.

Jeremiah & Chris:

Transformed SBP-TBP-ExV

Colonies were too numerous to count (TNTC)

Justin and Dafne

Transform ligation of SBP-B12 insert into expression

TOP10 competent cells were used

July 12

Michelle:

Check transformation of SBP (Spe1 and PstI)-LRP (PstI and XbaI) digested with XbaI and PstI + EP on AMP plates.

PCR colony check 24 colonies with Forward primer (Control promoter) and Reverse primer (Terminator) and ran gel.

Jeremiah & Chris:

Colony PCR of plate #1

No positive colonies or failed PCR

Justin and Dafne

Colony PCR check of colonies produced by SBP-B12 in expression plasmid transformation

Colony check successful

Culture successful colony in LB-amp overnight

July 13

Michelle:

PCR check colony #9 from transformation of SBP (SpeI and PstI)-LRP (PstI and

XbaI) digested with XbaI and PstI + EP with Forward primer (Control promoter) and Reverse

primer (Lysine antisense) and ran gel to double check if PCR colony #9 is truly successful.

Cultured 2 new colonies from 7/10 KAN plates containing successful transformation of SBP (SpeI and PstI) and LRP :::PstI and XbaI) with TB-KAN broth.

Jeremiah & Chris:

Colony PCR of both plates again

Culturing colony 1-9 from plate #1 and colony 2-4 from plate #2

Ligation of SBP-TBP into constitutive vector – transformed on 07/15

Justin and Dafne

once OD of the over night culture reached 0.6, L-arabinose was added in varying concetrations

Tube 1: 0.001% Tube 2: 0.01% Tube 3: 0.1% Tube 4: 1.0%

Control: SBP-B12 expression plasmid not induced by L-arabinose

After 4 hours, time sample 1 was pelleted and placed in -80 freezer

After 8 hours, time sample 2 was pelleted and placed in -80 freezer