Experiments This Week
Monday – Ran two RNA 15% pre-cast gels of last Friday’s o/n reaction and imaged.
Tuesday – Cas9 presentation; data suggests rtcB is not ligating the substrate; planning, reading and organizing day
Wednesday – ran a 15% pre-cast gel of a 1 hr ligation reaction (5th). Increased the amt of unlabeled substrate by 10x. Forgot to include the “no rtcB” control. Seriously?? Even so, data is very indicative of the protein not working. Setup one more o/n ligation reaction (6th, 3rd with new substrates). Stored RNA stocks in -20C and -80C.
Thursday – Ran a 15% pre-cast gel of the o/n reaction. Data confirms that rtcB is not ligating. Hybrid substrates arrived. Started a 1L culture of Keio delta rtcA, to see if I can get it to grow to OD600 = 0.6 by 7 p.m. but it did not. Allowed it to grow o/n at 30C as a very large o/n culture for protein expression on Friday.
Friday – re-expressed rtcB from Keio delta rtcA::amp. Harvested pellets and froze in -80C.
Purchases for the week = $69.08
Tentative Schedule for Next Week
Monday – protein purification and nanodrop; if have time, attempt to run gel to confirm protein; pour some 20% SDS-PAGE RNA gels and store for tomorrow; make stocks of the hybrid substrates; start an o/n ligation reaction with the RNA substrates and new hybrid substrates; meeting w/ Khalid at 4 p.m.
Tuesday – store hybrid substrates at -20C and -80C; run gel from Tu o/n reactions; stain gel w/ SYBR gold (note, might be good to do only one of these reactions at a time, to ensure that when I run the gel, the bands do not dissipate; running two RNA gels at once last time seemed detrimental). Potentially start another o/n reaction.
Wednesday – run another gel of the substrates; double check all notes and organize data
Thursday – depending on what the gels show, meeting with Dr. Weinert
Friday – TBA
Check and re-check your controls.
Working over a flame is always a good idea.
Using the cold box instead of ice has really helped speed things up.
Don’t get stuck in the details. Also examine what the majority of the data is suggesting. Not every band has to make sense.
You do not need duplicate lanes if the signal is clear. Think about why you’re doing something, rather than just doing lots of something.
Don’t forget the proper way to store RNA. Look up later what kind of tubes to store it in. Cryovials probably aren’t the best choice.
Never use the really old centrifuge in Dr. Weinert’s lab. It takes forever to spin down.
See if I can use one of the VWR CryoPro boxes from the Weinert lab or if not, order a pack for myself. That will make things so much easier.