Team:Nevada/Week 14

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Week 14: August 20 - August 25

Contents

August 20

Joe:
gel 167 - RFP pcr good, gel purify
Digestions: SBP(SpeI/PstI), RFP(XbaI/PstI)
Ligation of two digestions
Michelle:
Western blot transfer continued from 8/17 for samples of small colony from transformation of M2-BL21 developed :::membrane.
Justin and Dafne:
Large scale expression (200ml) of L-arabinose induced SBP-B12 protein with 5 nM vitamin B-complex
Jeremiah & Chris:
Western blot protocol (BL21 cells with no thiamine added)
Run time points on a polyacrylamide gel (BL21 cells with thiamine added)

August 21

Michelle:
Western blot transfer of remaining supernatant from last Western blot transfer of small colony from transformation :::of M2-BL21.
Justin and Dafne:
Obtain protein from cells as done before
Purify protein using Ni-NTX nickel column (per protocol)
Analyze using western blot and coomassie brilliant blue
Jeremiah & Chris:
Western blot development (BL21 cells with no thiamine added)
No band appeared after development so IPTG promoter could be the problem (picture on iphone)
Cancel other expression attempt using IPTG and added thiamine
Attempt to express our gene by use of a TET promoter
Set up phusion PCR for the TBP+ gene

August 22

August 23

==August 24==