Saturday 10/27/12

10:30A – 8:ooP

  • Stop O/N confocal by 11A.
  • Confocal data check: Late positions start too low.  Not sure why — maybe middle position to close to edge changed microscope positions?
Fly Work
  • Collect virgin Espl[D]/ sim[D].  
  • Flip fly stocks (3/4ths complete)
  • Flip fly cages, 12 noon
  • some larvae hatched on both Psc and Pc plates (not 100% penetrant knockdown at least. Now to see if we get any phenotypes at all…)
  • 5PM, collect virgin 
Testing knockdown:
  • Fix Pc and Psc knockdown embryos
  • Fraction of embryos saved at -20C
  • rehydrate embryos, block, O/N incubation in m anti-AbdB
  • Fly cages continuing to lay excellently.
DNA FISH probe making
  • spin down and miniprep colonies.  Plasmid Concentrations look good (250-350ng/uL)
  • Check O/N probe synthesis run.  No short probes detected.  General band in 647: either loading dye or un-encorporated nucleotides.
  • Run test reaction with new 5kb plasmid DNA.
  • Testing reaction on gel. — Typhoon not talking to computer.  But looks okay on Sapphire gel box.
  • Out of dGAC nucleotide mix for labeling reaction.  Got old frozen 100 mG GTPs from Sara.  Making new dilute nucleotide and nucleotide mix solutions.
  • Set up O/N DFISH probe synthesis.
  • Fix S2 cells on coverslip.  Prepping for DNA FISH with small volume.
  • DNA FISH labeling at 37 O/N with 25uL of probe between coverslip and slide.  (we’ll see if this can be removed and washed without detaching the cells…)

RNA FISH in culture cells, troubleshooting

  • Fix cells in 8 well slide
  • Treat in 100% MeOH at room temp 6 hrs, then O/N at 4C.  (permiabilize/dissolve membranes to allow better probe penetration).


Snail studies

  • Confocal 41 positions of MP10 sna/y.
  • top/bottom looking good, still on position 2 (good 10 sections of clearance above and below out of 60 section .31 um steps).
  • not looking on track to finish all 41 before 1PM tomorrow, increased scan speed to 7, dot resolution still looks pretty sharp on the 780.
  • now going at about 3 per hour, positioning still looks okay.
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