9:00 am – 10:40 pm
Live imaging
- live imaging of telomere-mmaple3 construct on STORM2
- need to correct live drift so we’re stuck with auto-correlation for these, no need to acquire beads
- blinking pretty well in current settings
- 800 mW set point on 561, used at 40% power on AOTF
- 2% constant 405 after first ~2000 frames to let the background bleach down a bit seems to give good blinking
- slowly ramp to 3% 405 by the end of the movie (60,000 frames)
- flipped out to 2D (conventional spots look better)
- autocopying to STORM2
- cleared disk space on STORM2 C
- Bogdan will take the analysis of these from here
Fixed imaging
- imaging ANTC-G6 in WT cells
- settings
- flipped back to 3D
- MEA buffer (5uL) no COT, fresh MEA in MEA dissolving buffer, fresh Glox.
- staining looks a bit dim/low contrast, maybe should have washed longer.
- temp might have been a bit low as well (75 instead of 78 — the glass actually cools down the block).
- Blinking looks good, hopefully still enough localizations for a robust characterization of domain size and overlap.
New stains
- anti-CycA on formamide treated WT cells from 8/11 fix.
- anti-Ph on WT and Ph KD cells from 8/11 formamide treated
- primary antibody 1.5 hours
Hi-C analysis
- working out intermixing model and illustration
- see Chromatin/Figs/Revision
- isolation of blue domains from flanking sequence for all domains not too robust