9:40 A – 8:00P, 9:15P — 11:40P
Fly Work
- collect virgin 2x HbZ.
- clear MTD x Pc1 parental generation.
- check Espl x sim crosses
write to Maria to cancel Wed sectioning
test gel PCRs
- No substantial bands
- New gel: Espl-DNA sim-all7, E1, E2, E1+E2, then sim-DNA and same 4
- E1 = primers L1,3-5 + R2,8-13. E2 =primers L2,6-8, R1, 3-7
- 5 min elongation, using BioXAct — running very low, should order more.
- Gel also fails. Some bands but all they match for both sim and Espl.
Polytene chromosome: Antibody confirmations:
- coated coverslips with sigmacote for preperation of polytene squashes
- made fix mix 1 and 2 for polytene squashes
- Lacking Lactic Acid for LAA lysis solution. Try with just acetic acid?
- Need liquid N to seperate coverslips
- Select fat wandering 3rd instars, move to H2O, rinse, then PBS.
- Learned to successfully identify and dissect out salivary glands (could use a bit more practice still, but with a few larvae I can get it). Following this protocol
- Still need practice with the squash — my cells are mostly separated out but the nuclei are mostly intact. A few nuclei are torn apart. Very few of the polytene chromosomes are properly separated though. Might require correct lysis solution (and more practice). Let’s wait until the lactic acid arrives.
- Starting staining on coverglass anyway. Incubate in SSCT and prep for DFISH.
- Incubate at 90C ~15 min (water bath in humidity chamber is slow to heat up. Hopefully this was enough and it works for these fixatives…). cent probe at 1:500 in DFISH hybe.
- Incubating O/N at 37C in prboe.
STORM analysis
- Bead analysis — bad calibration curves, causing most molecules to be class 9 in vis channel
- Including class 9 still allows molecule matching to <20 nm in XY and <40 in Z (losest in a while).
- H3K27me3-647 and Pc-488 look pretty close. Rb 750 not working, need new antibody… –> Ordered abcam antibody to try.
- STORM didn’t work well of K27, Pc. Should prep in sealed coverslip, reduce drift issues, get better buffer. See if we can find flatter sections and get good 488 switching next time.
Cell culture:
- Plate cells
- switch primary culture to new stirring flask.
- 330 ng/uL is about 10 pmol per uL for 50 bp DNA.
- dilute 1:100 in DFISH hybe buffer, add to cells.