Thursday 03/15/12

9:45 A – 9:30P

  • minor z drift issues still observed in STORM run.
  • Time to take vertical beads for z-fit.  at 1:1000 density is a bit low. try 1:500
  • using ND 2-3 for z-fit bead images. Might be saturated?  Need to check — intensity meter in hal software saturates at 10,000.  not sure if this is in raw 16 bit integers or something else.
  • Rinsing out antibodies on embryo stain to test new Hybridoma bank antibodies.
  • clean coverslips: 2 new batches of 30 mm slides
  • label 100 nm polystyrene beads: TMR (red) label seems to have worked, looks fine in turnkey.  elute 2 and 3 should contain beads and limited loose dye.
  • attempt 4 color label: FITC, TMR, Cy5.5, IR800.  5 uL of each (except IR800, only have 5, used 2.5).  with 20 uL beads in 100 uL reaction.
  • New fly cages: YW and MP09 (from collection plates)
  •  New fly plates. 5 min microwave too long for 600 mL .  Try 4 min.
  • Gel extract PCR cloning.  Concentrations low – 4 ng / uL (used to get 20-40).  Maybe very old gel extraction kit and ddH2O pH drift.  In theory should still be okay for ligation.
  • Attempting ligation of Taf1 and aTub84 cloned fragments 3uL PCR product/insert: 1 uL of pGEM Teasy in 20 uL reaction.  30 min at RT followed by 4C O/N.  Transform tomorrow.
  • Start work on ACS fellowship app (due April 1st.  Need to get paperwork to Matt by Monday).
  • New antibodies: dk a-m Cy3B, dk a-shp Cy3B, dk a-Ch Cy3B, dk a-Ch 750.  Still need to record AB peaks (data is saved just not measured).
  • Mount slides of stained embryos from hybridoma bank.  Let cure O/N.
  • O/N confocal of s04_engrailed (stain looks awesome, need to remake these probes, all more recent attempts look horrible).  Started setup at 5P.  Imaging at 1024×1024 for speed.
  • Run initialized at 7P.  Too much dead space above embryos in offset, restart (with same offset, -6um) fixes extra dead space.  Looks like another software glitch, first 3 embryos z-positions from run don’t agree with quick video.
  • amp plates available from Stephen for tomorrow.
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