2:45p – 7:15p
Review
- revising introduction of models
Cell culture
- clone8 cells growing fast. A bit overgrown, need to split this afternoon.
- separated ‘non-adherent’ fraction of Kc167 cells remain primarily non-adherent. Adherent fraction of remains adherent.
- test plate Kc167 cells from new NA fraction and top fraction of previous adherent plate (2 coverslips each)
- test plate 4 wells of clone 8 cells on polyL coverglass
- all cells adhere excellently, density may be even higher than optimal / intended.
DNA paints
- Rinse out stains from embryos, (65C)
- O/N staining of embryos with BX-C-405 (2:30) + cy5 secondary probes (1:30). Probably should dilute secondary down.
- Rinse out AbdA stain of previously-Ubx labeled S2 cells (stained since Friday night). Call this sample UA
- coat 415 amine beads at 1:5000 onto UA coverslip, crosslink these onto cells (5 min).
- no sign of beads (washed of after post-fix? No free amines left on outside of cells to crosslink?)
STORM analysis
- Had commented out the write infoFile part of splitQVdax. Rerunning just this part on Ubx and AbdA data so we can open the dax files split and analyzed so far and start calibrating fit parameters.
- Ubx splitting completed ~14 of 17 movies in ~24 hours.
- Analyzing STORM movies for Ubx and AbdA data (not all movies complete, will need to rerun later. Fortunately I have avoid / skip overwrite defaults so this should be easy).
- set all bead analysis parameters using InsightM with 0 overlap search and no drift correction.
- set all DaoSTROM analysis to 2d with 6000 frame correlation drift correction.
Prep
- construct new PDMS STORM chamber (last one PDMS finally delaminated from glass & epoxy. I wonder if this was a side affect of adding the COT buffer (certainly correlational).