10:00 A – 7:00P, 8:30P – 12:40A
File maintenance
- Alistair12 out of disk space! Black-DNA region run crashed due to running out of disk space
- STORM analysis crashed due to no free disk space to save files
STORM analysis
- Fab7 3x cy5 512×512 looks interesting, switching may be a little slow on this view. Running through analysis software. This also results in massive files.
- phM-FLAG on Alistair10 has good images consistent with other data on PhM, but the 512×512 view wastes a lot of space with only one nuclei visible per field of view (800+ GB).
Sectioning
- humidity supposedly reset to be low last night
- new humidity monitor reading steady 55% still. — moved to STORM2 for comparison.
- cleaned and coated coverslips (5x gelatin chromium mix), double batch
- sectioned en-labeled yw embryos. ~14 coverslips.
- cleaned and coated coverslips (5x gelatin chromium mix), double batch
Probe making
- test gel of temp and high primer conc. High primer conc looks good, band strength is higher and higher with more primer.
- Let’s try fewer rounds of PCR (e.g. 30) less room for junk to be made and grow (also another difference between clean runs like my emPCR / some of my small scale PCR, Jeff’s PCR’s, and the regularly dirty ones.
Cell labeling
- dissolve Tertiary-405 in 100 uL hybe (~30 nmol)
- nanodrop Tertiary-405 >5000 ng/uL. (this has 10 bp overhang. Re-ordered version hasn’t arrived)
- nanodrop full-length-secondary 3′ cy5 ~ 1,800 ng/uL (check)
- 3 uL Ter-405 (nanodrop ~5 ug/uL) + uL Sec-cy5 1.8K ug/uL + 80 uL hybe buffer split 4 ways
- samples = Blue-P1, Yellow-P1, Black-P1, no-primary control (first round of probes)
Meeting with Ajaz
- layout figures (see sketch)
- to do: put together STORM fig1
- planned: repeate original imaging