Failures update

Fly Genetics

  1. Failed knockdown:

    • 2x Mat alpha tub driving any of the TRiP valium 20 or valium 22 PcG target shRNAs.
    • Gives viable F1 and F2s or infertile F2s. No PcG phenotypes.
    • Kill all current crosses
    • Kill all expanding Gal4 lines
    • Kill all shRNAs except stock vials
  2. Missed emergence of Df/Esc,CyO virgins

    • Kill all bottles except one. (expand Esc/Gla)
    • (First round Esc crosses / collections going better than expected).
  3. Failed recombineering:

    • Espl can’t recombine due to inversion.
    • Kill all ongoing Esc crosses
    • Kill all expanded Esc bottles and vials

Failed Staining ultra cryo sections

  1. AbdB, Ubx staining followed by 5min PFA/GA post-fix then BX-C oligoPAINT

    • Weird background patterns of rings from slide drying out.
    • Forgot DAPI (useful for locating sections).
    • No BXC clusters found – staining failed.
    • Sections highly warped.
  2. AbdB+m-cy3B O/N staining, post-fix then BX-C oligoPAINT 8 hrs during day:

    • small upstairs oven takes hours stabilize temperature — big failure in switching from 60 to 42C. Cooling it down to RT, turning it off, and it still wants to run back up to 60C long after its been reset to 42C.
    • All channels failed.
    • high Cy3B background in yolk tissues.
  3. O/N simple and straight BXC

    • No staining
    • 55C 25 min hot wash too much for tissue sections?
    • need better protocol version management
  4. RNA FISH on ultracryo sections

    • Could not find sections on Turnkey to check staining
    • Saw sections previously on cell-culture phase-contrast scope
    • Sections irrecoverable. Slide tossed.
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