9:40 A – 7:00P, 8:20P – 10:45P
- Flip collection cage, 9:45A. Keep plate to make new bottles
- Pick up more 10x TAE and 20x SSC from stock room
- Picked up 6 new pints of 100% EtOH.
- Found issue with K27:H3 ratio. Fixed = much cleaner data!
DNA FISH probe making
- Spin down BAC7 DNA probes (slightly colored pellets. sample 2 (‘false start’) has more blue color than sample 1.
- resuspend in D-FISH hybe buffer
- run 5 uL on a gel
- No probe — sample not substantially digested.
- Ordered new DNA pol I and DNAse I components from Promega following JW protocol recommendations.
- Test synthesis with regular DNTPs and a range of DNAse concentrations (2,4,6,10,14 uL in 50 uL rxn). All with 1 ug of BAC 7 DNA.
- Running test gel: Bands from original DNA all visible. Possibly slightly fainter at higher DNAse concentration but not dramatically so.
Embryo labeling
- rehydrating embryos
Fly work
- Flipped MTD x Pc, MTD x Psc, and MTD x Scm into new bottles. Embryos/larvae growing well.
- Flipped sim[D] into 2 new bottles. Should be ready for collection cage next generation
- Flipped yw Act5-Gal4 into new bottle.
- Moved sna[18], Sp/CyoZ and Pr/HbZ into bottles. need to start building double-balanced sna[18] again (previous one died and Mounia hasn’t been able to do it).
Literature
- Fraser et al ascribe bursting to transient association into transcription factories. http://www.nature.com/ng/journal/v42/n1/pdf/ng.496.pdf.
embryo fixation
- Thawing DFISH mix
- # PM Fix embryos
- # Block 1 hr
- # stain with rb anti-Pc O/N at 4C
Results to date on K27me3 dynamics
- found bug in analysis of K27me3 to H3 rato, now fixed (?).
- Fix image 5 nuclear mask (others look okay);