Experiments This Week
Monday – prepared additional buffer C for protein purification; pHed to 8 on ice; made overnight culture of Keio delta rtcA::km/pQE70-rtcB::amp and grew at 37C in Lynn lab
Tuesday – Expression of Keio delta rtcA Shuman and Raines cultures. Both cultures failed to grow in a reasonable amount of time. Made more overnight cultures and grew at 37C in the Lynn lab.
Wednesday – Expressed Keio delta rtcA::km/pQE70-rtcB::amp Raines culture. Used fresh ampicillin from the Conticello lab. Expressed about 2.5 hrs (0.5 mM IPTG) and froze in -80C. Made an overnight of Keio delta rtcA::km/pQE70-rtcB::amp for Shuman expression.
Thursday – Grew Keio delta rtcA::km/pQE70-rtcB::amp cells until OD600 = 0.6 and expressed with 0.1 mM IPTG o/n 16 hrs at 17C (Shuman). Used fresh ampicillin from the Lutz lab.
Friday – Purified Raines and Shuman expressions using new Ni:NTA beads. Had to clean the Ni:NTA column with 0.5 M NaOH and 20 min sonication, to get out the junk from the contaminated Shuman beads. Afterward, had no more problems getting it to flow properly. Eluted rtcB with 300 mM imidazole and dialyzed overnight with a stir bar at 4C in 1L buffer C w/ DTT freshly added.
Saturday – changed buffer C w/ DTT added the day before each hour three times. Afterward, checked concentration of each isolate by Nanodrop. Shuman = 1.68 mg/ml and Raines = 0.77 mg/ml. Flash froze 100 ul aliquots in liquid nitrogen and froze at -80C. About 170 tubes total (~90 Shuman, ~80 Raines).
Purchases for the week = $38.96