Saturday 04/12/14

9:30a – 4:00p

Computer Management

  • attempting to give users rights to share their hard-drives on the network without having full Admin capabilities to do anything they want.
  • Tried adding my ‘User’ account to these groups
    • “Performance Monitor Users” — can access performance counter data locally and remotely.
      • This doesn’t actually let me launch task manager without Admin rights. Not too surprising, since I can kill tasks from task manager.
      • Can’t even access the windows program “Performance Monitor” without Admin rights.
      • Can’t launch the “Resource Monitor” without admin rights either
    • “Access Control Assistance Operators” — windows / google seem to provide no information on what this really does.
  • Can now open preferences, still get asked for admin password to try to change advance sharing file permissions

Chromatin Project

Data Processing

  • SplitDax finished (hopefully for the last time) on E02 data
  • running bead averaging / compression on E02 data
  • DaoSTORM finished on E03 data

troubleshooting hybes

  • have staining now of C01 in both new cells and old cells
    • looks better in new cells
    • neither is comporable to earlier stains or restained c8 cells.
    • some cells have strong/decent stains and some cells lack staining.
    • pretty sure there’s higher background, different quality. May relate to lack of RNase treatment in new cell round. —

NewCellDecentSTORM NewCellDecentSpot PrevGoodC08cellC01restain C08C01_matchedLaser oldCellNewBuffer

More notes

  • should have checked — C01 is 240 kb Y. We probably do have mRNAs in cytoplasm.
  • don’t have spots in every cell, when we do have spots the cytoplasm is dimmer. maybe I’m just staining RNA. Can treat these cells and see if we lose stain?
  • previous C01-C08 batch of cells were first imaged 1 day after I made new RNase solution, so I don’t have it for sure in my notes but I’m pretty confident these guys didn’t skip the RNase step. So current stains are consistent with not denaturing the DNA, except that a side by side re-treatment of C08 cells does denature for strong incorporation. So something is still different in the cell prep that affects the denaturation, rather than the denaturation itself.
  • maybe RNA also dilutes out concentration of local probe (I kinda doubt it, probe concentration is pretty high since whole hybe soln is visibly blue).
  • maybe somehow cells are over-fixed and won’t denature? I think I’m very consistent though on fixative and fixation times…
  • I guess I often let them sit longer in the glycerol than the 30 min I have been doing — I sort treated that as a minimum to get properly equillibrated rather than an exact time step.

To try

  • RNase treat current stained C01 new cells, see if foci are lost. Treatment started 1:20 pm.
  • RNase treating new cells prepped with new buffers. Will then do extended pre-hybe.
  • 95C denature of RNase treated cells prepped yesterday and restain with C01.
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