Team:Nevada/Week 13

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Week 13: August 13 - August 17

Contents

August 13

Joe:
Primer stock- RFP f-anti
PCR of [f]SBP-promoter to RFP anti
Michelle:
Transformation of Friday’s ligation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested with
SpeI and PstI with Terminator (PstI and XbaI) and Promoter (TETrbs) (EcoRI and SpeI) into
Top 10 competent cells onto KAN plates.
Miniprep colony M2 from transformation of PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) followed by PCR ::::purification with glass milk because of mistake in miniprep protocol.
Nanodrop miniprep of colony M2 PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) before transforming into BL21 ::::competent cells and onto AMP plates.
Justin and Dafne
Culture successful colonies from last weeks ligation in LB-amp until OD reaches 0.6
Add IPTG in varying concentrations
Tube 1: 0.01 uM Tube 2: 0.1 uM Tube 3: 1.0 uM Tube 4: 10.0 uM
After 4 hrs, pellet, place in -80 freezer
After 8 hrs, pellet, place in -80 freezer
Transform SBP-B12 with L-arabinose promoter into BL21 cells
Jeremiah & Chris:
Miniprep colony 2
Transform purified plasmid into BL21 expression cells
Attempt to express the protein while in TOP10 cells using an IPTG gradient
Took a 3 hour and overnight time point
Jeremiah & Chris:
Miniprep colony 2
Transform purified plasmid into BL21 expression cells
Attempt to express the protein while in TOP10 cells using an IPTG gradient
Took a 3 hour and overnight time point

August 14

Michelle:
Check yesterday’s transformation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested
with SpeI and PstI with Terminator (PstI and XbaI) and Promoter (TETrbs) (EcoRI and
SpeI).
Transformation yesterday of colony M2 from transformation of PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) on :::AMP plates was wrong. Needed to be transformed onto Ampicillin-Chlorophenicol (AMP-CM) plates.
Re-do transformation into BL21 competent cells onto AMP-CM plates.
Justin and Dafne:
Culture SBP-B12 in BL21 cells to an OD of 0.6
Add concentrations of L-arabinose as done previously, also adding 5 nM Vitamin B-complex
Take time samples as done above
Jeremiah & Chris:
Run the time points on a polyacrylamide gel

August 15

Michelle:
Check transformation of colony M2 from transformation PCR Fusion SBP (SpeI and
PstI)-LRP (PstI and XbaI) with BL21 competent cells on AMP-CM plates.
Culture smallest colony from this transformation, which we will call M2-BL21 with LB AMP-CM broth.
Obtain pellet and supernatant preparation from 8ml of colony M2 from transformation of PCR
Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) not IPTG induced for Western blot transfer.
Justin and Dafne:
Perform western blot analysis of both protein expression experiments
IPTG unsuccessful, L-arabinose induced SBP-B12 in BL21 cells very successfull
Jeremiah & Chris:
Western blot protocol
Cultured BL21 colony from transformation plate

August 16

Michelle:
Western blot transfer continued for pellet and supernatant samples of colony M2 from
transformation of PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) not induced with
IPTG and membrane developed.

August 17

Jeremiah & Chris:
Developed western blot of expression in TOP10 cells
No expression was detected (took pictures of the membranes on my iphone)
Attempt to express the protein while in BL21 cells using an IPTG gradient
Took a 3 hour and overnight time point
Cultured same BL21 colony with thiamine supplement added
Michelle:
Western blot transfer of 8ml from culture to obtain pellet and supernatant samples of smallest
colony from transformation of M2-BL21 from 8/15.
Jeremiah & Chris:
Run time points on a polyacrylamide gel (BL21 cells with no thiamine added)
Attempt to express the protein while in BL21 cells using an IPTG gradient (BL21 cells with thiamine added)
Took a 3 hour and overnight time point