Wednesday 01/09/13

10:00A — 10:45P

Cell culture

  • blocking IF first.  
  • secondaries for IF first (2 m-750 (6:1), 6 wells m-488 (2.0))
  • 10 min in .1% GA, 4% PFA post fix.  wash in PBT, then 2xSSCT
  • following Brian’s protocol.  Need rubber cement.
  • O/N FISH first batch incubated with lid on.  Previously this led to mixing of probe.  Might have mixed centromere probes AACAC and AATAT.  should be okay for the purposes of this experiment.  Try to keep separate for IF first batch.
  • DFISH first wells O/N in PBT, ready to image.
  • Immuno first wells O/N at 42C.

Probe making

  • spin down precipitate samples
  • set up digestions: 6 tubes ~ 70uL each, 5 hr digest
  • Precipitating DNA O/N.  will run gel tomorrow

Confocal O/Ns

  • MP10 cc13s.  Imaging O/N on 780
  • MP08 cc13s try 2.  – assembled all MP08 slides from past year (7 slides).  Very few non-LacZ cc13, even fewer mitotic phase.  Last slide has a few potentials, will have to complete mRNA counts to see if we can distinguish.
  • one slide imaged through on 780 between 4p-10p.  second slide imaging O/N on 700 (by eye, these embryos look better).

Fly Work

  • cross remaining shRNA lines to MTD (Ph-D, Ph-P, Pc3).  Should have saved more shRNA SCM males to cross to extra MTDs
  • Crossed 5 more lines of potential rescues to parentals for complementation screening.
  • several of the potential rescue lines in serious need of flipping
  • re-ordered Dorsal / daughterless / twist flies without Mat tub drivers (since we got driver from Max)
  • virgin collection finished.
  • Flip yw cage, 9:30P — laying pretty well (might just have been holding eggs since original bottles were old though).  Might try fixing tomorrow.   Will need to make more fly plates very soon.
This entry was posted in Summaries and tagged , , . Bookmark the permalink.