10:00 A – 6:30P, 7:50P – 10:20P
Embryo work
- Flip collection cages ~10:00 A
- No hatching from Mat apha tub x Rad21, Scm, Pc(3), Psc(1) shRNAs.
- all these embryos appear unfertilized. Frequently moderate deformaties to egg shape, substantial variation in composition.
- Not expected M/Z phenotype — something else is going on with these shRNAs. Giving up other shRNAs for now.
Fly Work
- Espl[ry1] deletion won’t recombine because its also an 3R inversion. Should use Df(3R)Bsc751 — nice compact deletion, kills just E(spl) cluster + Gro.
- 3R between sim and Espl has a ~3 centiMorgan/Mb recombination rate and 11 Mb distance — should have ~33% recombination, should be an easy double mutant. (maybe make Scm, Pc at same time?)
- Tossed all previous Espl[ry1], sim[D] crosses.
- shRNAs aside from scm not working, all tossed except stock lines (can make one more try with 2x mat-alpha tub vp16). I suppose I could try with 1x first? Just some small test crosses for viability at 29C?
STORM
- Failed:
- Very high background due to slide drying out with protein/dye/DNA bound.
- Need to rinse slide repeatedly but always keep hydrated!
- No sections flat enough to image.
- No DAPI counterstain to help locating images.
Embryo ultra-cryo sectioning staining
- ID slide with good embryos using DAPI screen of coverglass. Locate on 20x air first!
- Blocking, combine primary and secondary antibody O/N at 4C (shouldn’t be penetration issues with exposed ultra-cryosection nuclei.
- Be sure to keep slide hydrated at all times. (also good for morphology).
Literature
- Pho maternal effect allele disrupts eve patterning (publication)
- is eve up-regulated in PcG mutants with weak en phenotypes? (eve is an indirect negative regulator of en: InteractiveFly)
- Maybe both get derepressed and one TF system wins. Whereas AbdB trumps all other TFs and gets to turn on as long as Pc is gone? Hard to explain stronger derepression in Pc mutant combos though.