Team:Nevada/Week 14

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