Monday 01/14/13

9:30 A – 7:30P

snail imaging

  • copy data from confocal (55 GB file).  Run O/N at speed 7 using GASP detector
  • projecting data from false start.  Delete lsm file (mostly blank, and the critical data has been copied into a .mat file).  Delete blank images.
  • Out of space on local drive D.  Moving confocal data 2012 to G: (GRAID) drive.
  • # start processing data

Steven lab meeting (see notes)

STORM Analysis

  • Initial run of dot-fitting complete.
  • start writing mini-viewer / interactive viewer for dao-STORM?
  • K9me2-750_AATAT-647e

    K9me2-750_AATAT-647e

    HP1-488_AATAT-647e

    HP1-488_AATAT-647e

    HP1-488_AATAT-647d

    HP1-488_AATAT-647d

    HP1-488_AATAT-647c

    HP1-488_AATAT-647c

    HP1-488_AATAT-647a

    HP1-488_AATAT-647a

    HP1-488_AATAT-647b

    HP1-488_AATAT-647b

    HP1-488_AATAT-647

    HP1-488_AATAT-647

    Hp1-488_AACAC-647_DIFSH-1st_drifting

    Hp1-488_AACAC-647_DIFSH-1st_drifting

    K9me2-488_AATAT-647_DIFSH-1st

    K9me2-488_AATAT-647_DIFSH-1st

    K9me2-750_AATAT-647d

    K9me2-750_AATAT-647d

    K9me2-750_AATAT-647c

    K9me2-750_AATAT-647c

    K9me2-750_AATAT-647b

    K9me2-750_AATAT-647b

    K9me2-750_AATAT-647a

    K9me2-750_AATAT-647a

Cell culture

  • 500 kb probe in situ day 2
  • hot wash 15 min, 60C
  • appreciable general nuclear background, no detectable specific signal.
  • attempting second round of 60C hot wash, see if background cleans up at all.
  • 17 min post wash at 60C in SSCT, after brief RT stint in PBT on microscope — background dropped percipitously to nothing.  possible weak dots visible in cells.
  • Rinse in PBT, block, label nuclear-lamina Cy3B.

Probe making

  • Order unlabeled primers for amplification of LC library sequences.  
  • mixing DNP RNA labeling mix:  250 nmol dUTP, want 3.5 mM solution.
    • 250 nmol in 1 mL is 0.250 mM.    want 250 nmol in 71.4 uL.
    • 7 uL GTP + 7uL CTP + 7 uL ATP + 4.5 uL UTP + 250 nmol DNP-11-UTP, fill to 70 uL.
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