3:30P – 11:55 P
Fly work
- Flipped most of rescue crosses. Several vials extinct. Should still have more vials than I want to screen though.
- New crosses of SCM males to MTD virgins (2 vials).
- Examine current crosses of all shRNA lines to MTDs — not looking good, food pretty dry (despite humid 30C). Moved to RT. Most vials from Monday’s crosses now have (a very few) pupae. Flipped these into new vials.
- Thursday’s crosses will need to get flipped later this week. Should screen vials for dead embryos.
Planning
- Ordered rubber cement from Amazon (should arrive Thursday).
- confirmed previously ordered primers are correct. Best to check staining of 500 kb probe before starting new round of synthesis for en?
Confocal
- Imaging MP05 embryos. Selected 44 positions (60GB)
- drift after 5 sections imaging sessions are starting inside embryo epethilium instead of above embryo field of view. cancelled and restarted.
- Imaging MP07 embryos for width measurements
Cell labeling
- coat coverglass with .5 mg/mL concavlin A (just add briefly and remove and add to next coverslip). 2 coverslips coated.
- Plate with S2 cells, allow ~1hr to attach
- Fix cells (over-fixed, ran 20 min). 4% PFA in 1/2 x PBT.
- incubate coverglass 5′ in 2x SSCT in coplin jar
- incubate coverglass at 60C in small hybe oven in coplin jar. (skipped 2.5 min at 95C step, which is to be done before this).
- Let cool to RT. Add probe to clean, dry glass slide, gently drop slide on top. Incubate O/N at 42C.
Data Analysis
- Running preliminary STORM analysis on Cajal. Using parameters optimized for last 3D HP1 dataset in S2 cells. Currently running all analysis from image 1 (should probably push this back a few thousand for some images, though we can also do that retro-actively with STORMrender so it’s not a problem).