Team:Stony Brook/Notebook/Week10
Week 10
7/31:
• Ligation of tagged hybrids with GST and SUMO vectors
• Transformation of GST tagged and SUMO tagged hybrids into DH5α E. coli
8/01:
• Made small scale liquid cultures of GST tagged hybrids
• SUMO hybrids → transformation failed
• Wanted to start protein purification, but our columns and Ni-NTA resin had not arrived yet.
• Samples sequenced properly!
8/02:
• Transformation of MBP LnqZQ, MBP LnqZA and MBP LnqZE into BL21 E. coli
• Miniprep of GST tagged small scale liquid cultures
• Nanodrop of minipreps
8/03:
• Columns and Ni-NTA resin arrived so we can start protein purification!
• Freeze-thaw cycles 1& 2 on the frozen cell pellets (GST LnqZ, GST A53, GST Epi)
• Small scale liquid cultures of MBP LnqZA, MBP LnqZE, and MBP LnqZQ
8/04:
• Finished the last freeze-thaw cycle for GST individual cell pellets
• Started the protein purification process by equilibrating columns and nickel resin with lysis buffer
• Followed all steps outlined for protein purification under the “Experiments and Protocols” tab.
• Large scale liquid cultures of MBP LnqZA, MBP LnqZE, and MBP LnqZQ, induced with IPTG
• Centrifuged large scale MBP liquid cultures, collected cell pellets
8/05:
• Nanodropped our elutions of GST protein purification products
• Since we did not get good results (most likely no protein), we did not nanodrop our elutions for GST Epi.
• Decided to run a gel in order to determine whether any protein was being purified even though nanodrop did not yield results
