Thursday 04/27/14

Posted on March 27, 2014 by admin

10:15a – 12:30a

Goals

  • Data processing
    • copy C05 and C06 data to hard-drives
    • Launch analysis of C03-C06 data
    • plate, fix, prep and hybe new cells
  • Probe making
    • RT reactions
    • test gel
    • probe purfication
  • cell staining
    • wash out probes
    • image multi-color cells on STORM2

Chromatin Project

Probe making.

  • finished making 16 new probes (2 failed, no template).
  • gel looks pretty uniform for concentration
  • F03 has weird series of bands — probably not good. Should remake. F05 failed completely (no template, I guess I knew this. Good negative control).
  • attempted 500 pmol scale synthesis. Incorporation looks less good, should stay at 400 pmol per 10 uL RNA.

Lib2_F01toG08_probes
F01-G08probePlots
F01-G08probes

Cell staining

  • fixed and prepped new cells for staining, including 1hr+ RNase A treatment at 37C.
  • F03-p3 2 uL, F02-p1 7 uL
  • F05 P3 1.5 uL, F06-P1 6 uL
  • F06 P3 2.5 uL, F07-P1 6 uL
  • Damn it I used the failed and questionable probes in the staining. Need to check these things!
  • Why do I get better yield on the failed probes?

Imaging

C07 on STORM2

  • 3 uL COT, 5 uL BME,
  • looks reasonable
  • Need to remember to take beadfields in the morning! Otherwise there will be lots of beads to manually parse out of ChromatinCropper.

C08 on STORM1

  • 2 uL COT, 5 uL BME
  • spots identifiable in all cells, but weak / small / low contrast.
  • realigned back-optics (way off center)
  • tweaked dual view to separate channels Cy5Cy3 3D. Alignment is off by 10s of pixels between channels.
  • Need to remember to take beadfields in the morning! Otherwise there will be lots of beads to manually parse out of ChromatinCropper.

Coding / Research Organization

  • established GitScripts repo on github, pushed scripts. Now these and their revision histories backed up somewhere. Yay!
  • pulled latest version of script databases into GitScripts and committed and pushed up to Github (This takes a couple commands, including a move command to get new scripts to there appropriate folders. I bet I can write a matlab wrapper to do this in a single click.)
  • added private protocols to GitScripts
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