10:45 A – 8:10 P, 9:00 P – 10:00 P
STORM
- 488 wasn’t bright enough to see single molecules at all last night
- try with completely fresh glox, some switching events detectable in nucleus. Alexa488 not as bright as Atto.
- 84% 488 laser power, 60% AOTF, harder TIRF (14.4) now gives more usable switching rate detection rate.
STORM analysis
- DaoSTORM gets confused by 3D out of focus feducials. Need to track with insight with higher threshold (or maybe DaoSTORM with higher threshold or single fitter). If using Insight, need to set max displacement to 0 so molecules don’t all get linked, making drift tracking impossible.
Genomic region selection
general goals:
- 2 5-15 kb regions yellow, red, blue, green, black
- 2 40-60 kb regions yellow red blue green black
- 1 90-150 kb regions, yellow blue green black
- 2 250 – 500 kb regions green black
Update
- selected 40 regions, largely from and surrounding ANT-C, BX-C and En.
Mouse
- HoxD region: chr2:72,327,275-77,059,774
sample prep
- stain cells for Hao: 647 + 750 and 647 alone