Tuesday 08/28/12

9:30 A – 7:00PM, 7:45P – 12:00A

  • 20 min etch Pc-647 sample.  Turnkey inspection looks over-etched, most embryos quite sparse.
  • Forgot that previous MP07 samples have Draq5 not DAPI.   Extracting nuclear masks manually through inverting DAPI channel and using custom nuclear rescue script cyto2nucmap.m (Image_Analysis) to back out nuclear masks.  mp07a and mpo7a2 affected.
  • STORM imaging of Pc-647.
  • Fedex hard drive to mounia
  • STORM4: pc-647.  750 Pc-GFP and Dm01 switching gangbusters
  • some background in 647, 10,000 frame bleach?  True signal is much brighter, should use higher threshold.
  • additional observations on Mazo Paper (TrxG and PcG Proteins but Not Methylated Histones Remain Associated with DNA through Replication, Cell (2012)) discussed by email with N Francis (see gmail archive).
  • STORM imaging comments: A few placques of resin, identifiable by non-bleaching nature and visible in all channels.
  • Analyzing more snail mRNA count data for promoter and enhancer modifications.
  • m124B308 position 11 on fail to export properly in imviewer_lsm.  mp07b positions 13 to 20 fail to export properly.  Loading into Zen 2011 Black for tif export and run through renaming script.  Import of both files into Zen less than 50% complete after 40 min.  Check again at 10:30P.
  • Substantially less switching in later movies (calibration movies show strong switching in 750 for 30-50,000 frames, run movies by 11PM are starting strong but have mostly finished switching by 20,000 frames.  This is going to be BAD for drift correction (as well as data storage).  Should shorten tomorrow.
  • Yuan et al 2012 Science claim dense histones recruit PRC2 and make Su(Z)12 active.  mononucleosomes not sufficient, piece of H3 is sufficient.  Propose nucleosome coated genes collect active PRC2 and thus compaction before methylation.  Also claim using nuclease accessability that compaction happens before K27me3.    So, if you kill the PREs and then the promoter, the nucleosomes can all come back.  Does the gene get K27me3?
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