Thursday 11/23/12

10:30 A – 7:30 P

Thanksgiving Fly work

  • expand YWs
  • Flip stocks
  • check crosses.  double HbZ not yet emerging.  recombineering crosses looking bleak, we’ll see in anotehr 5-6 days…

Probe making

  • New library stats
  • spin down dissolving probe.  Gently Pippett off supernanant (pouring easily lets some agarose fragments from pellet into the new solution).
  • Add 2 mL fresh ddH2O and incubate at 37C shaking another ~2 hr +.
  • Butanol extract DNA probe
  • In EtOH precipitation at -20C O/N.
  • Tomorrow: Precipitate and resuspend in DFISH hybe

Cryo-slide labeling

  •  Coverslip 1-2: Hot wash 55C for cent-labeled slides (10 min?)
    • hot washes coverslip-on heat block does not work well — solution whicks off the top of the block and also evaporates rapidly.
    • Much better to use hybe oven and keep slides on styrophome pedestals in the custom made humidity chambers.
  •  Coverslip 3: Aspirate and save primary antibody (a-Pc + a dm01) from slide
    • Rinsing in PBT
    • Block 1 hr
    • Add secondaries
    • check staining on Turnkey — looks good all channels (didn’t add 647 channel yet).  Should add H3K27me3-647 direct label.
    • O/N in PBT at 4C.


  • Aspirate off and save primary antibodies from well B.  Rinse,
  • secondary stain
  • Wells G: Aspirate off restain primaries (K27, K9), Rinse, wash,
  •  reblock and secondary stain well G.  See old primaries are still there / can be relabeled?
  • 6:2 750 is also rabbit spots, though label on side and ratio indicate that it is an R&D systems antibody.   Will confirm with no-primary control channel next.
  • definitely background spots in G 750.
  • rabbit 750 spots much weaker but still detectable (again through 405) in 8-well B.  Good staining of antibodies but insufficient pre-block means high background.  Should block for 90 min (20 min not good enough).  Probably should have sodium borohydride treated these cells as well.

Confocal (4P-8P)

  •  multiplex cent-DFISH + H3K9me2 (and others) seems to work.
  • should be able to get better stains with SB and more block and more rinses (backround could be lower).
  • Interesting/suggestive colocalization patterns already.  Maybe we should also stain some polytene cells.
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