10:30 A – 7:30 P
Thanksgiving Fly work
- expand YWs
- Flip stocks
- check crosses. double HbZ not yet emerging. recombineering crosses looking bleak, we’ll see in anotehr 5-6 days…
Probe making
- New library stats
- spin down dissolving probe. Gently Pippett off supernanant (pouring easily lets some agarose fragments from pellet into the new solution).
- Add 2 mL fresh ddH2O and incubate at 37C shaking another ~2 hr +.
- Butanol extract DNA probe
- In EtOH precipitation at -20C O/N.
- Tomorrow: Precipitate and resuspend in DFISH hybe
Cryo-slide labeling
- Coverslip 1-2: Hot wash 55C for cent-labeled slides (10 min?)
- hot washes coverslip-on heat block does not work well — solution whicks off the top of the block and also evaporates rapidly.
- Much better to use hybe oven and keep slides on styrophome pedestals in the custom made humidity chambers.
- Coverslip 3: Aspirate and save primary antibody (a-Pc + a dm01) from slide
- Rinsing in PBT
- Block 1 hr
- Add secondaries
- check staining on Turnkey — looks good all channels (didn’t add 647 channel yet). Should add H3K27me3-647 direct label.
- O/N in PBT at 4C.
Cell-labeling
- Aspirate off and save primary antibodies from well B. Rinse,
- secondary stain
- Wells G: Aspirate off restain primaries (K27, K9), Rinse, wash,
- reblock and secondary stain well G. See old primaries are still there / can be relabeled?
- 6:2 750 is also rabbit spots, though label on side and ratio indicate that it is an R&D systems antibody. Will confirm with no-primary control channel next.
- definitely background spots in G 750.
- rabbit 750 spots much weaker but still detectable (again through 405) in 8-well B. Good staining of antibodies but insufficient pre-block means high background. Should block for 90 min (20 min not good enough). Probably should have sodium borohydride treated these cells as well.
Confocal (4P-8P)
- multiplex cent-DFISH + H3K9me2 (and others) seems to work.
- should be able to get better stains with SB and more block and more rinses (backround could be lower).
- Interesting/suggestive colocalization patterns already. Maybe we should also stain some polytene cells.