Research week 16
Monday - Reintroduced Ian to running a 12% SDS-PAGE gel of proteinase K digesting rtcB. Wrote out a protocol for the part I couldn't show him while I was doing lab TA. He finished it no problem and stained the gel stain overnight; I finished re-designing the DNAzymes and substrate until I was satisfied with them; went to the first TA lab session and listened to Mr. McCormick
Tuesday - finished last details of the DNAzymes and ordered them with the substrate (remember to ask Steve what the final cost of this was for my records). Destained the rtcB gel and realized that I had not told Ian the correct protein concentration for him to use - it was too small.
Nanodropped a new set up my FAM RNA substrates and calculated the concentrations. Made new FAM RNA, 10mer 2 RNA, phos 1 (hybrid), sub 1b (hybrid) and 20mer hybrid stocks. Setup a hybrid ligation reaction with Raines and Shuman protein at 37C overnight. (Accidentally included the same volume of Shuman protein as Raines.)
Wednesday - poured and ran a 15% SDS-PAGE gel, filtered, under RNA conditions of the hybrid ligation reaction (using tips from the Dunham and Conn labs). Stained with SYBR gold for 50 min. Destained twice 15 min each in !x RNA TBE. Showed Yue how to use the fluorescence instrument in biochemistry - she helped me get into the building. Results: 10mers did not show, but both Raines and Shuman rtcB show product of the same size as the 20mer bands in buffer and in reaction.
Thursday - took time to catch up on inputting scheduling into Google calendar for classes, study and read a few short papers
Friday - finished the EPEX training and paper work for fastlane access for NSF proposal, with the help of magical office person Stephenie Thioubou (this took a long time). The new RNA substrates arrived!! Put them in cold room for storage until Monday. Ian repeated the rtcB digestion with proteinase K at higher protein concentration and left the gel staining. I destained it and let it sit on my benchtop over the weekend. Hopefully allowing it to go through only one 20 min destain and not leaving it shaking will be ok for that long - I think, based on listening to Yoshie before, that it should be ok.
Weekend Plans - study bioorganic reaction mechanisms and do problems; work on NSF proposal; read the papers I've been printing out, along with Dr. Weinert's papers
1. On my 15% gel, Shuman rtcB was loaded more than Raines, due to me forgetting and not accounting for the concentration.
2. Next time, make sure to image the gel at a PMT that does saturate band intensity so it can be properly quantified. Use ImageJ to quantify bands in the Raines paper this weekend.
3. Lanes still show a strange smearing pattern, but it's different from the FAM substrate smearing, interestingly.
1. Measure the activity of rtcB on an Au NP.
2. Measure rtcB ligation in presence of the two DNAzymes and substrate - is ligation product produced and if so, what kind?
3. Does rtcB need GTP for the reaction? (Look this up over the weekend.)
4. What is the reaction rate of the DNAzymes (forward/reverse)? At what temperatures? metal concentrations? pH? How does this rate compare to Kevin's DNAzymes? Do the fewer hairpins in the construct increase the reaction time? If so, by how much?
More questions coming soon....