9:20 A — 7:15P, 8:15P – 10:00P
- Check polytene cell staining. Faint glow of nuclear laminin visible. pretty dim, does not have ring shape, see top and bottom (would be easier to distinguish as diffinitive stain by confocal).
- Rinse in PBT and try -20C acetone permiabilization
- polytene cells have bacteria, just like cell culture
- bacteria are actually in the blocking solution (confirmed by direct imaging of Hoechst labeled block. Can see aggregates dividing and swimming populations). oh no! Might be responsible for antibody issues lately. Need new block urgently.
- Colenso offers 100% donkey serum, recommends use at 10%.
DNA FISH in embryos
- hot washes at 37C in post-hybe solution
- check samples on confocal
- DNA is degraded, yw? embryos must have been left out too long at RT
- Stain aborted
- Restarting DNA FISH with 1-10 hr DFISH fix MTD embryos. (need to fix more wt embryos)
- Prehybing in 2x SSC 50% formamide at 37C
In situs (SCM)
- Day 1. 3 tubes, stain for en, AbdA, hb, and eve, mRNA, En and AbdB protein.
- Digest remainder of 50 kb probe, running O/N
- Abcam antibody arrived, make rb-750. Looks okay, 12 uL dye -> 1.7 dyes/antibody, .3 ug/mL
- F1 to R2 900 bp fragment. Looks good on Gel, band not present in yw. see if we can optimize conditions to get a stronger band, this is much weaker than AbdB control band.
- cut out band for digest.
- Screening Espl: 24 combinations + 24 controls. Strips have primers L1, L4, or L7. Each tube has primers: R12, R13, R11, R4, R10, R7, R8, R9 (moving progressively longer)
- Running 12 min elongation with longAMP pcr O/N (annealing temp 63C).
- sort / collect virgin 2x HbZ AM
- PM virgin collection 2x HbZ
- 7 vials of complementary crosses of 2x HbZ