Wednesday 12/05/12

9:30 A  – 7:40P, 9:00P – 12:00A

Confocal dot distances

  • Finish O/N confocal run of Ubx-AbdA data

Probe making

  • sent order to LC Sciences for chromatin types
  • On why previous probe might have failed:
    •  over drying? — DNA and RNA break down when overdrying.
    • claim cold precipitation unnecessary, hang over from older protocols.  This at least matches recent report in PLoS one comparing DNA prufication kits at different temperatures and EtOH (more EtOH is good, temp no signficant effect).
    • reference
In situs
  • rinsing out primary antibodies

Cell culture

  • Check staining.  Looks promising
  • blocking, will add antibodies – Dm01-405,750, H3K9me2-488
  • stained
  • confocal imaging — some cells have very suggestive dots, but quite few cells.
  • Staining has probably failed.

Fly Work

  •  split MTD large cage into too (too dense, not laying sufficiently well).
  •  set up small cage of Su(Z)12,MTD F1s.  — skipped, these embryos are starting to hatch and crawl after all.   Maybe Pc1 will work better?
  • collect larvae in buffer for dissection.
  • # set up snail double balancing crosses generation 2
Polytene chromosome 
  • Dissect and mount polytene cells
  • attempt mechanical chromosomal spreading.  Some cells at least appear well spread, might need to repeat.  Could attempt on poly-L coverglass instead of poly-L slides.  
  • Label with centromere stain.  Left in 90C too long (30 min?).  And dry.
  • slide #2 labeled with DAPI, and then with H3K9me2. No staining.  Dm01 works well.  Try H3K27me3 tomorrow.  If this fails, look up protocol?


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