Experiments This Week
Monday – prepared additional buffer C for protein purification; made up 1 L RNA 10x TBE; 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. Shuman cells failed to grow inexplicably. The Raines culture I expressed at 37C instead of 32C. Discovered that ampicillin used was expired. Had to scrap the experiments and cancel Weinert incubator. Made up two more overnight cultures.
Wednesday – Expressed Keio delta rtcA::km/pQE70-rtcB::amp Raines and Shuman cultures. Isolated Raines rtcB and expressed Shuman rtcB w/ 0.1 mM IPTG o/n for 16 hrs at 17C in Weinert incubator. Washed my Ni:NTA columns and made notes on ligation reactions.
Thursday – purified the Shuman and Raines rtcB cultures according to Dr. Weinert’s advice. Shuman lysate spilled on cold room bench during 1 hr incubation, but scooped it back up with pipet. Was significantly dirty. Did extra washes on it. Xin in Weinert lab showed me how to do dialysis. Used 10 kDa cut off dialysis bag from the Lutz lab. Dialyzed protein with three buffer C changed, one per hour, and the last allowed to dialyze overnight with a stir bar (forgot the stir bar for the others).
Friday – Concentrated the rtcB in a 15 ml Amicon filter, but it both samples precipitated. Saved the pellets to show Khalid on Monday.
Purchases for the week = $94.65