10:15 A — 11:15 PM
Polytene cell staining
- hot washes in 2x SSCT (45 C in coplin jar in dry oven)
- check staining — cent probe looks good (rather small foci though)
- Antibody staining H3K9 did not work. Should have included Dm01-Cy3B to check antibody penetration for this tissue prep.
- attempt to permiabilize with MeOH, 10 min at RT. Relabel with DM01-Cy3B (O/N at 4C). (Probably just succeeded in removing cent-probe + Hoechst without sufficiently permiabilizing. Abcam recommends doing this step at -20C. Could try acetone at -20C for 10 min if this doesn’t work.
Cell culture, 50kb probe test
- hot washes
- check staining –> no dots 🙁
- not enough probe? Try again, higher concentration of probe
- restaining at 1:10 probe
- cent probe
- failed 50kbp from cell culture.
- missed virgin collection last night
- collect males and females for later sorting from 2x HbZ crosses
Group meeting to prep: 12/17
Revisions for snail paper.
- calculate probe concentrations (100-250 ng/uL stocks)
- Add cell boundaries to Figure 1
- Compute total mRNA counts for competitively labeled probes.
- Send to ML
- primer order:
- SimD-dna: AbdB, F4R1, F2R2, F1R3, F3R3, F2R1, yw-DNA: AbdB, F2R1