Tuesday 09/24/13

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
  • smallRegions4_nano
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