Alistair's Lab Notebook

9:30 am – 11:30 pm

To do (Oligo Secondaries)

• review new fig 2 layout for Brian
• send additional images of spots for Fig 14 for Brian

Sequential staining

• validated that toe-hold probes do work, competitive probes do not work, (for displacing oligos).
• F06 high concentration cocktail kind of works, not nearly as bright or as many localizations as I expect though for this fairly large region
  • (30K frames is all I got).
  • Maybe F06 P3 probes work better, I seem to have a lot more batches of these.
• Imaging F06 regions. (just a handful of short movies, so that I can test the washout and BXC staining today too).
  • keeping 405 laser below 10% max power.
• ah, second hybes really not working (at least after treatment with competitive probe. Though I have trouble believing that is the problem.
  • Should try again with 1st hybe on scope.
  • should try again with just bleach no toe-hold probes.

Datasets

• 14-07-04 data:
  • rapidly gets too sparse sampling: (buffer rundown?)
  • consistent with superposition of F06 and BXC (both P3 probes)

• 14-07-02 data
  • much of this data is also rather sparsely labeled, especially given the contrast at brightness of the spots
  • this data also shows clear signs of buffer run down, images 7 on notably fewer localizations
• chamber will need to pause and flow fresh buffer more frequently for the long term imaging this way.

Chromatin To do:

• compute total number of loci imaged in final data set
• compute total number
• compute number of genes covered

Internal domain analysis

Blue data

• With region size having such a weak effect for large domains on total domain area (for internal regions), we really need to define the medians very well in order to make good fits. This will require substantially more data than we needed for the complete domain scalings
• new regions to do:
  • F03toF04
  • F04toF05
  • G01toG03
  • G01toG04
• should also look at small pieces of BX-C using new library

Deeper analysis of Y-internal domain data

• E06 data pretty noisy
• E07 data rather sparse (just 3 images on each of 2 different systems)
• E06E07 data is actually quite good, but needed to do better on censoring the out of focus images. — elongated x-y blurry blobs no good.
• new yellow regions to do
  • E06 (rpt, lower conc)
  • E07 (rpt, lower conc)
  • E08toE09
  • E07toE09