9:45a – 9:00p
- Attempted to swap memory into Cajal.
- Cajal won’t boot up with any of the memory
- restored Tuck to 6x 8GB DIMMS.
- Cajal has 12x 4GB DIMMS
- wrote to IT to help get system back up.
PH project: requests from Ajaz
- pick some poly-lysine tonight around 6:45pm?
- Below are the things which need to be adjusted for main model figure. We need to reorganize the model figure (no. 6). In this figure we need
- two schematics for models
- plot showing fraction clustered as a function of PH-ML conc.
- Representative images
- Plot showing diameter varying as a function of PH-ML conc. OR distributions of cluster diameter at different conc. of PH-ML as you plotted for low, mid and high PH-ML concs. (Here I wonder if we show plot for cluster size vs PH-ML conc. we might need to run simulations to show how diameter changes as we increase the PH-ML conc. Something similar to what we have for fraction clustered vs PH-ML conc. plot)
- Please see Bob’s comments regarding this figure also. (in the manuscript revised by Bob) According to him schematics are not easy to follow and simulated lines should be smoother (which I think means running the simulations many more times)
- Actually let’s put this on hold. Currently redrafting paper into shorter 4-fig version and I don’t think there will be time or space for the modeling.
- still should run PSC clustering data.
- run oligo-clean and concentrate of probes
- quantify probe concentrations
- Re-measured temperature of hot-plate.
- setpoint 88C, read with block inverted, thermometer on top, now 78C. Used this for hybes.
- setpoint 85C, read with block inverted, thermometer on top, temp 71C.
- setpoint 88C, read with block right-way-up, embedded thermometer, 81C
- setpoint 85C, read with block right-way-up, embedded thermometer, 78C
- C07 treat with 2x SSCT 50% form along with other cells when they come out of RNase. Restain with E04 probe. Denature at usual 85C (glass temp ~78C).
- new cells RNase treated, extended prehybe at 47C. stain with C01. Denature at usual 85C
- new cells RNase treated, extended prehybe at 47C. stain with C01. Denature at 100C (glass temp will be 95C at best)
- Aim / hope: RNase treatment decreases background from expressed locus, extended 47C prehybe increases suspetability to melting during denaturing step.
- hope 2, change in hot-plate as source of problem now fixed?
- old cells, RNase treated, stain with new E06 probe (1.5 uL of 2,000+ng/uL probe + .5 uL primary)
- old cells, RNase treated, stain with new E07 probe (3 uL of ~800+ng/uL probe + .5 uL primary)
- working on methods section for paper
- sent current working draft back to Brian.
- finished checking equations, found small error in my calculations. Still don’t match authors calculations.
- finished writing up review comments and submitted review.