11:00a-4:30p, 8:00p-11:00p
Goals:
Cell culture
- plate cells on Poly-Lysine coverglass (with Bogdan ?)
- passage remaining cells (with Bogdan ?)
- Prep 9/2 plate and new plate for DNA FISH.
Staining
- O/N primary staining of Pc and anti-flag.
Probe making
- RNA cleanup on controls for Bogdan
- check RNA samples for last set of probes made (e.g. F6). Use RNA for straight RT reaction.
STORM
- Finish O/N STORM of F6
- test STORM of E6-405
- test STORM of F6-405
Fly work
- Flip fly lines — not managed.
STORM
- Finish O/N run on F6 data, 11:15a (not all cells complete but data from last few runs looks good)
- 2D movies, with beads tracked in separate channel using quadview. Conventional images contain z-scan.
- should attempt more 3D movies. Requires calibrating PMT offset on STORM2 again.
- Transferring data to Monet.
Cell culture
- dilute Kc cells 1:2 and plate media (600 uL each) onto poly-lysine treated UV irradiated coverglass.
- Extra media goes to passage cells into new flask. After cells settle, remove extra media/unattached cells, provide fresh media.
- ordered more Schneider’s Drosophila media for culture.
- Fix cells, 10 min 5% FA in PBT
- DNA FISH prep these cells. Move all the way to 50% formamide. Toss previous 9/2 fixed cells.
Probe Making
Black regions
- RNA prep controls for Bogdan using 1x rinsed plates. Really weird large, tight 240 nm peak.
small regions
- take: 1, 4,5,6,7, 9,10,11 on to oligo-cleanup and T7 reactions
- actual IDs:
- (1) E02 13 kb green (between R/Y)
- (4) E06 Epc (12 kb YELLOW)
- (5) E07 Tou (15 kb YELLOW)
- (6) E09 RED 8 kb intronic tRNA locus, in between blue
- (7) F02 BXC_Y_left (15 kb)
- (9) F09 BLACK 18 kb region next to RED1
- (10) F12 Taf1_(17kb yellow/green)
- (11) G01 LabRegion_(Lab_Zen2) [83 kb but missing piece of ANT-C]
- Nanodrop results