9:45 A – 9:30P
- minor z drift issues still observed in STORM run.
- Time to take vertical beads for z-fit. at 1:1000 density is a bit low. try 1:500
- using ND 2-3 for z-fit bead images. Might be saturated? Need to check — intensity meter in hal software saturates at 10,000. not sure if this is in raw 16 bit integers or something else.
- Rinsing out antibodies on embryo stain to test new Hybridoma bank antibodies.
- clean coverslips: 2 new batches of 30 mm slides
- label 100 nm polystyrene beads: TMR (red) label seems to have worked, looks fine in turnkey. elute 2 and 3 should contain beads and limited loose dye.
- attempt 4 color label: FITC, TMR, Cy5.5, IR800. 5 uL of each (except IR800, only have 5, used 2.5). with 20 uL beads in 100 uL reaction.
- New fly cages: YW and MP09 (from collection plates)
- New fly plates. 5 min microwave too long for 600 mL . Try 4 min.
- Gel extract PCR cloning. Concentrations low – 4 ng / uL (used to get 20-40). Maybe very old gel extraction kit and ddH2O pH drift. In theory should still be okay for ligation.
- Attempting ligation of Taf1 and aTub84 cloned fragments 3uL PCR product/insert: 1 uL of pGEM Teasy in 20 uL reaction. 30 min at RT followed by 4C O/N. Transform tomorrow.
- Start work on ACS fellowship app (due April 1st. Need to get paperwork to Matt by Monday).
- New antibodies: dk a-m Cy3B, dk a-shp Cy3B, dk a-Ch Cy3B, dk a-Ch 750. Still need to record AB peaks (data is saved just not measured).
- Mount slides of stained embryos from hybridoma bank. Let cure O/N.
- O/N confocal of s04_engrailed (stain looks awesome, need to remake these probes, all more recent attempts look horrible). Started setup at 5P. Imaging at 1024×1024 for speed.
- Run initialized at 7P. Too much dead space above embryos in offset, restart (with same offset, -6um) fixes extra dead space. Looks like another software glitch, first 3 embryos z-positions from run don’t agree with quick video.
- amp plates available from Stephen for tomorrow.