Wednesday 04/11/12

10:00A – 11:30P

  • coating 5o mm coverslips
  • put coverslips in metal, vertical mount holder.  Don’t stay balanced in ceramic holder.  Need to alternate positions or the coverslips stick.
  • Do coating in 400 mL in 1000 mL beaker (not 300 mL in 600 mL beaker, metal rack does not fit).
  • Sonication steps need to be done in square glass case (only thing that fits holder and fits in sonicator).
  • Ultra-sectioning HP-6, H3-750 sample.
  • stuff growing in SOC.  explains colonies in control ligation possibly. Try transforming again…
  • repeat transformations using all new pipettes and new sterile tubes, new SOC.
  • gel extracting PCRs 2,3,5, 8 (no well/gel-box combo capable of 50 wells).  Largest is 8 20 uL wells, so we extract 4.
  • concentrations of Dfd2, AbdB-1, AbdB-3, and iab5E all look good: 12-20 ng/uL.
  • Ligating O/N at 4C with 2 uL plasmid: 1 uL PGEM.
  • cut sections: 3 slides of H3-750, Hp1-647.
  • 4 slides of H3-647, Hp1-750.  ribbons not holding together well.  humidity low and sections curling in and swirling with extra static electricity.
  • working on journal club presentation on Li and Pirrotta 2011 paper.
  • Fixed Pc-GFP flies 15 min fix, prepped for oligo-STORM
  • hybridizing with oligo-paint Cy5 probe.  embryos translucent in hybe, very hard to see in small volume PCR tube.  Denatured probe and embryos separately, 5 min, then together 3 min (at 94C), then dropped to 37C.  Embryos at RT while heat block drops temp.
  • Oligo paint probe 50 picomoles in 50 uL.
  • Select small number of embryos to mount after quick hybe rinse at RT, 2x SSC dilution, then mount.
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