Thursday 05/29/14

11:00a – 12:45a

(9:00a-11:00a, volleyball practice).

Meeting with Bogdan

  • see notes
  • Bogdan Data: \cajal\AlistairTemp3

Chromatin Data Analysis

Chromatin Cropper Coding to do

  • integrate radius of gyration computation
  • integrate flip h/v/transpose options for conventional images
  • fix auto-loading of conventional images (currently not loading 000n conventional images correctly, instead loads first image from movie stack).

Troubleshooting

  • implemented flip h/v/transpose (FlipImage, now in ImageTransforms in matlab-functions)
  • this is not actually the problem, it is just the name matching that is the issue.
  • missing some conventional images for the L3 F03-p1, F02-p3. Just find spots from STORM images (the contrast is good enough for these).

Analyzed data

  • saved data in C:\Users\Alistair\Documents\Research\Projects\Chromatin\Data\2014-04-23_Lib3data2clr

Data that needs attention

  • E02 data staining a little weak (and many section in bad focus).

Sequential Staining and Imaging

  • set up objective heater and bio-optics chamber
  • objective heater movable collar broke (seems to have happened after inappropriately loosening the screws on the side instead of the back of the unit
  • black spacer broke off. Epoxied this back on.
  • removed tiny hex-screws holding down top to expose mechanism. Plugged in loose side of collar and resealed with set-screw. Device now fixed!
  • heating objective (should have started this earlier, takes 1 hr+).
  • 5:45pm, started incubation in secondary. 1:100 dilution of 100 mM stock into 50% formamide 2x SSCT, diluted a further 1:4 into hybe dilution buffer.
  • Imaging sample Lib2, F03-p1, F04-p3
  • cannot find S3-A647 secondary (supposedly ordered on 12/18/13). Also cannot find P2-A405 supposedly ordered at same time.
  • proceed with S3-A750, just do sequential multi-color :(.
  • ordered new primers, 5′ A405 for P2 and P4, 3′ A647 for S2, S3, and S4.
  • both sequential hybridizations look excellent.
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