9:50 A – 7:00P, 8:30P – 11:20 P
- Clean and coat coverslips
- washout fix from O/N fix samples, move to EtOH
- rehydrate, check for presence of staining.
- Respond to team about NSF BIRS proposal
- Pol I arrived
- 4:00 PM check samples at 70C.
- 6:20 PM, remove samples (18.5 hrs baking)
- Confocal: check stains. All look good. Epc-dig 514, inv,tou bio looks especially promising. Dfd tub also looking good.
DNA Probe making:
- New enzyme mix, screen variable concentrations of high DNAse solution
- Running test gel, (15uL rxn).
- seems to be an issue with template DNA — having difficulty detecting band from template DNA.
- 3 of the tubes detect decent DNA (not red-label). These are now magenta labeled.
- Transform new competent cells with 5kb probes: AbdA, Ubx, Antp, toll, SxlA, y. Using old amp plates (I overload the amp and the control plates from the last round of old plates looked okay. We’ll see if we get the right plasmids back…).
Select DNA regions for STORM of chromatin colors
- Download color maps, format in Galaxy into BED files uploadable to UCS browser
- transcribed genes from my probe set:
- yellow (has PolII and Pc along full length S2 cells). Blue chromatin in Kc cells
- sog, vnd, Kr, kni, tup, pnr, sim, eve(paused) = Pc repressed BLUE chromatin.
- sna, ind, hb, Delta, Toll(paused), hbr(paused) =black chromatin
- hbr paused in S2 cells (black). upstream of Neu3 active 120kb region
- Mes2 = very ON PolII (Red)
- Mes4 = okay ON PolII (Yellow)
- Neu3 = okay ON PolII (yellow/black/red in Kc). Inside 120kb mostly very active transcription region.
- Rho = okay (stalled and ON?) yellow
- cact = okay (stalled and ON?) red
- neur = okay (stalled and ON?) red/yellow/blue
- Notch = okay (stalled and ON?) red/yellow/blue
- tkv = okay Pol II, some good spots (Pc/red)