Monday 10/19/15

10:00 am – 6:50 pm


  • send info to Stanford Dev Bio.
  • finish and submit application to Cornell Physics (due today)
  • write to XZ about new experimental design.
  • synthesize lots more dsRNA
  • start new KD of Ph
    • experiment with 2 day KD, 3 day KD, daily serial KD,
  • update website


  • dsRNA production
    • ran DCC5 column clean up on PCR products of ph-p and ph-d from last week
    • eluted in 20 uL and set up parallel 20 uL T7 reactions with 10 uL product each.
  • cell culture
    • passaged cells, adherent cells from Schneider’s media culture moved back to SFX — my previous flask of cells in SFX is at too low density.
    • cells are ready for more RNAi as soon as I have dsRNA.
  • probe production
    • ran new round of lib4 prep using 1:10 dilution of stock library. Samples C01 to C08 (with reverse primers G01 to G08)
    • this amplified sooner (nearing saturation at cycle 24 instead of waiting till after 30).
    • still have substantial amounts of large band product in all lanes except well 8. Well 8 was the last one to amplify.
    • still see rather high starting values broadly distributed (including 1 unpopulated well). suspect issue with QPCR camera. Should populate a different position on the plate next time.
  • also note my old ladder is completely degraded. current ladder on bench degrading a bit.
  • embryo labeling
    • treated embryo samples 5 min with .1 M HCl (previously skipped in failed labeling last week. BB thinks this step is important for labeling).
    • labeled E24 and E20-E24 on embryo sections (previously failed labeling last week).
    • incubated 3 min at 90C,
  • try staining sectioned embryos with Hb smFISH probes
    • using fresh frozen embryo section.
    • 1 uL probe in 30 uL hybe dilution buffer, incubating at 37C overnight.

new project planning

  • see protected post.


  • Sluis et al 2015 G&D: CRISPR/Cas9 induction of DSBs
  • Aymard et al NSMB 2014
  • Clynes et al PLoS ONE 2014 (nice immunoFISH)
  • Roukos et al, Science 2013
  • Iacovoni et al EMBO 2010
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