Wednesday 12/04/13

9:40a – 11:30p


  • show Hao new project 2 results
  • RNase-Away / bleach treat everything on my bench
  • fix and stain new RNA cells. Allow 1 hour prehybe.

Project 2

Chromatin colors, double stain prep

Probe organization

  • reorganized both boxes of completed probes.
  • Should start a new database in google drive to track completed probes, primer used, etc.

Region selection

  • List of regions to make for double multi-color staining:
    • G2, G3,
    • G5, G6
    • G1, G2
    • F11, G1
    • F12, G1
  • D12, F11, F12, G1, G2, G3, G5, G6, + neg control
  • Setting up PCR. Spec primers:
  • at 20 bp (common), 10 uM is ~66 ng/uL. 40 bp is ~132
  • dilute D12p is good, F12p is good, F11p is bit low, G01p 1:1.5 (add 50uL ddH2O to 100 primer), G02p add 0.8, G03p add 1.1, G05 add 1.4, G06p add 1.6;
  • set up PCR to run overnight

STORM analysis

  • analyzed G1 and G9 data (80 kb blue ANT-C piece and 75 kb freestanding blue).
  • G1 image quality degenerates over time (especially conventional images).
  • suspect bleaching by TIRF beam. Discuss arraying image direction with Bogdan.





  • E4 and E5 may not have worked. But then I was having issues with STORM4
  • F12 (Taf) definitely worked, very clean dots.
  • Imaging F12 overnight.

Cell staining

  • Fix new cells
  • prep cells for hybes
  • prehybe 4 hrs at 47C in prehybe mix
  • attempt RNA stains of D12, E08 and F06 (E10 basically empty). 3 uL D12, 1.5 uL of the others. 1 uL L2S1 (a647) in 20 uL hybe dilution mix.
  • incubating at 47C ~10pm.

Chromatin Colors Data Analysis

Bead Processing:

  • Running z-calibration for 647 beads for G09 data from STORM4
  • Running chromatic calibration for G09 data from STORM4
  • Analyze chromatic beads for G1

For future: Troubleshooting difficult probesets

  • Prep for sequencing:
    • good: G2, G3, D12,
    • oddly difficult: G4, F3, F4, F5
  • Wrote to Jeff to inquire about new optimized seq prep protocols.
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