9:30 am – 10:00 pm
STORM
- L2 F02 F03 — F03-p3-750 not very bright given size
- F02 stain looks like it could be better — a bit of background here too
- imaging L2 F02 F03 anyway, shorter exposure
- 9 pm set up imaging of L3 E08-p2-6 E09-p3-7
- not as bright as expected for these loci. Remade Glox buffer (6 days old now), spots brighter
Probe Making
- ChrLib4 probe synthesis A01-B12
IVT reactions
- 30 uL total :
- 10 uL buffer nucleotide mix
- 10 uL DNA template
- 1 uL RNAsin Plus
- 1 uL T7
- 8 uL ddH20 nuclease free
25 x master mix
- 250 uL buffer nucleotide mix
- 25 uL RNAsin Plus
- 25 uL T7
- 200 uL ddH20 nuclease free
RT
- 3 uL primer
- 2 uL dNTP
- 14 uL RNA
- 6 uL RT buffer
- 1 uL RNasin
- .5 uL Maxima
- 4 uL ddH2O
25x RT P2
- 75 uL primer P2
- 50 uL dNTP
- 150 uL RT buffer
- 25 uL RNasin
- 12 uL Maxima
- 100 uL ddH2O
6x RT P3
- 18 uL primer P3
- 12 uL dNTP
- 36 uL RT buffer
- 6 uL RNasin
- 3 uL Maxima
- 24 uL ddH2o
- tubes 1,3,8,17,19
cleanup
- froze samples, try plate-column cleanup tomorrow.
PH polymerization
- working on manuscript revisions / additional data analysis.
- quantify total overlap by fraction of localizations of one channel within localization precision (~25 nm) of other channel. I had done this for PH-WT and not PH-ML before. Quick estimate drawn from last cell of PH-ML data (20% PH-ML with PC and 30% PC with PH-ML). Should go back and compute all cells and average.
- quantifying distribution of non-co-clustered populations — this we basically had already, just plot the
~coclusteredmlists and run cluster size stats on those. - quantifying correlation of cluster sizes. — finished off analysis started yesterday. Approach uses an OR mask and then overlap to call clusters, so all localizations from two clusters that share a portion of pixels are counted — this reduces artificat from partial misalignment on the 20-50 nm scale due to residual uncorrected drift and residual uncorrected chromatic aberration.
Other Notes
- new GSA membership