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Team:Nevada/Week 14
From 2012.igem.org
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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
- Joe:
- Colony PCR - gel 171 right side both colonies good
- Michelle:
- Western blot transfer continued from yesterday for remaining supernatant sample for small colony from transformation :::of M2-BL21. Membrane developed.
- Cultured more M2-BL21 with LB AMP-CM.
- Jeremiah & Chris:
- Ran gel of phusion PCR products to confirm existence (gel #169)
- Purify gene using glass milk protocol
- Ran gel to see concentration (gel #170)
- Ligate phusion products together
- Expression plasmid phusion product already don
August 23
- Joe:
- miniprep of SBP-RFP cultures
- PCR - gel 173 SBP-RFP-Tet[f] bad, Tet[f] backbone good
- culture more RFP plasmid
- Michelle:
- Glycerol stock 1 ml of M2-BL21 + LB AMP-CM.
- Protein purification: Pellet 40 ml of M2-BL21 + LB AMP-CM, sonic pulse, follow procedure
- for Nickel (Ni) column to purify protein.
- Jeremiah & Chris:
- Trans formation failed due to use of the wrong cell line (BL21 cells instead of TOP 10)
- Attempt transformation again
August 24
- Joe:
- miniprep RFP
- new primers: RFPEPftanti50, RFPEPftsense50, RFPEPresense60, RFPEPreanti60
- SBPftsense, SBPftanti, SBPresense, SBPreanti
- PCR sample of each - gel 174 show all but RFPft good
- PCR purify
- DPN digest of RFP
- Michelle:
- Coomassie staining to detect purified protein, SBP-LRP.
- Jeremiah & Chris:
- Transformation failed again
- Purified fresh TET backbone phusion product
- Ran on a gel (gel #176) to confirm concentration
- Glass milk purify TBP+ phusion product gene
August 25
- Michelle:
- SDS-PAGE Coomassie staining of gel.
- Large-scale expression of protein purification: Cultured 1 ml of M2-BL21 glycerol stock
- into 500 ml of LB AMP-CM.
- Jeremiah & Chris:
- Ligated phusion products together (TET and TBP+)
- Transformed into TOP 10 cells
