9:00 am – 7:30 pm, 8:45 pm – 12:00 am
- mounting sample in the sharp-nosed conic capsules is actually excellent for ultra-cryo mount (should order more of these)
- 12:00 pm, incubating L7 and L8 on recently sectioned embryos.
- 5:00 pm, mounting sample into Bioptics chamber.
- apparently mounted incorrectly, no embryo sections visible in chamber
- 8:00 pm,
- confirmed some sections are still bound, though I notice even in this ribbon several embryos detaching
- other ribbons depostied on this coverslip detached completely. Maybe go back to trying gelatin/chromium again?
- 9:30 pm, remounted sample now correctly localizes embryos
- notes: cy3 channel autofluorescence not very bright. Should WGA-488 label embryos and use 488 channel for contrast
- accidently partially bleached cy3 dyes while searching for sample
- notable issues with sample depth and focus. Trust the focus lock, but remember there are sometimes two places where it starts to report signal. Also trust the 4x image.
- 10:38 pm, added bit 1 in R-buffer (with dextran sulfate)
- observation: adapter tubing didn’t transmit pressure with dextran sulfate in solution. had to add solution directly to chamber.
- My have introduced translation into stage, will need to check.
- incubating 30 minutes
- thin stiff tubing is very trick to pipette through and readily creates air bubbles
- thicker tubing pipettes much better. easy to remove air bubbles.
- cy3 spots look good
- Alexa647 spots detectable, but I expected better for 15kb.
- mapping out design for focus lock
- copied STORM2 layout, matched to parts list on STORM5.
- started new illustrator file
Checking sequences in L8. These are the readout probe binding sequences in L8, and the 20mer root of the corresponding probes.
These appear to be Stv8 through Stv1.
choseSeqs = unique(stvSecs,'stable')';
upgrading matlab to 2016a.