Tuesday 12/11/12

10:10 A

Check PCRs (in blocks 3A (sim screening) and 3B (Espl screening)

  • better way to do this: complementation test back to original sim[D] and Espl[D] lines.  Just need to expand those lines back to bottles, will need ~100 vials of each
  • Need to screen lines to ensure mutation is stable, not lost to double balancers.  TM2/TM6 flies are ebony, others should carry candidate E/S alleles.


  • DNA antibody (to think about) from Millipore.  Measure DNA accessability to antibodies in heterochromatic regions?

Working on lab meeting presentation

  • Figures to explain compaction assay, looking at ends

Fly work

  • new bottles for MTDs
  • Flip fly stocks (just rack 2.  Rack 1 flipped at end of Nov.  Ready to flip again after group meeting)
  • Discuss recombineering crosses with Sam Kunes.  Recommends backcross complementation tests with original lines.  Of course!  This is much better than PCR mapping of breakpoints.  Yay!  Need to expand sim[D] and Espl[D] lines again.
  • Expand Esc ?  (to test after break?)
  • New YW bottles (to set up cages after break)

Cell Culture

  • Move cells out of 29C incubator (Colenso has reserved for 70C)
  • 750 #2 405 signal looks bad.  Much stronger signal from 405-750 #1 (left most bottom).  Use this one for STORM
  • Both 488 750 double labeled wells no good.  488 and 405 signal very weak.
  • 488 #2 no good.  488 #1 very stong.  Both Cy3B wells very strong.

  • switch back to 256×256 view — swap in the 50 mm focal lens
  • updated thresholds for 647 and 750 STORM parameters files for Spot Counter
  • TIRF angle has changed dramatically.  13.25 good TIRF.  
  • 647 looks very dim in STORM (more like 750), low contrast.  Not sure why?  
  • # check 405 power
  • # bead slides
coverslip Ucryo staining
  • Found another coverslip that has some adhered (though only partially, no full embryo section is flat).  Attempting DNA FISH + Hp1A in embryos.  Prepping for DFISH.
  • Make new 20% dextran-sulfate in 4x SSCT + 50% Formamide (2X SSCT final conc.)
  • Sigmacote silconize slide.  hybe sections on slide O/N.  (Rinse off in coplin jar tomorrow).
STORM analysis
  • z-calibration files with substantial drift z-drift during calibration are impossible to use.  
  • Use previous passable z-fits, and run data on Cajal.  


  • # check with Alec about DSHB antibody order
  • # abcam antibody?
  • #
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