Tuesday 10/23/12

9:20 A – 6:30P, 8:00P-11:00P (remote)

S2 cell DNA-FISH labeling

  • Fixing S2 cells for DFISH (1/2x PBT 5% formaldehyde)
  • cells still attached, looks promising.
  • cells preincubated at 92C for 3min, 60C for 20min, moved to RT
  • probes denatured at 92C for <1 min (should be single stranded centromere probes denature not as necessary except for some secondary structure).
  • probes added to samples at RT, samples in humidity box in 37C room.
S2 cell RNA-FISH labeling
  • Fixing S2 cells for RFISH (1/2x PBT 5% formaldehyde)
  • cells in prehybe at 55C downstairs (in humidity box).
  • added Epc-dig, tou-bio, top row  150uL each
  • added Epc-dig, en-bio, bottom row 150 uL each
  • forgot to heat denature probe secondary structure.  we’ll see how much this matters.
Embryo prep for STORM continued
  • embryo labeling
  • washing out secondary antibodies from O/N embryo staining.
  • Pc embryos on final rinses
  • embryos in tertiary antibodies at RT, 12:00P.
  • mount Pc embryos for confocal (1:3oPM)
  • post fix Pc embryos
Fly Work
  • Espl x sim[D] pupa just beginning to darken. (start collecting virgins in ~2 days?)
  • TRiP knockdowns just beginning to emerge.  (Maybe ready for cages by Friday). 
Other prep
  • flipped fly cage
  • made new DFISH fix mix.  Aliquot and Freeze. 
  • Microscopy seminar: confocal
  • Write to Ting about S2 cell DFISH
  • Working on BJ review
  • Write to Mike about kinetics paper.
  • Write to Jacques, draft response to Mike. 
  • split S2 cells
  • Book 780 ‘scope time: Thur night, Fri night, Sat night and Mon night.  


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