Wednesday 07/16/14

10:00 am – 12:00 am

Sequential Staining

  • all at 37C (heated objective)
  • flow 2X SSCT to rinse out storm buffer
  • prehybe in 2x SSCT + 50% formamide, 15 min
  • hybe 30 min with S1toe5 (3uL in 1 mL hybe dilution buffer)
  • wash 15 min in 2x SSCT + 50% formamide
  • rinse in 2X SSCT
  • flow STORM buffer and image sample — all staining removed. So toe 5’s work.
  • rinse in 2X SSCT
  • prehybe in 2x SSCT + 50% formamide
  • flow hybe S3 (3uL in 1 mL hybe dilution buffer)
  • get DC power supply for heated chamber, heat to 37C (reading 28C at surface sensor).
  • hybe S3 3 hour.
  • wash out hybe 3
  • apparently 15 bp is too much to remove (Still don’t understand why the toehold doesn’t work in reverse here, though I guess this isn’t really a toehold situation, its a ‘slap away’ the invading strand — we have a 15 bp anchored duplex with a 30 bp tail guarding a non-homologous 30 bp of sequence that the secondary wants to bind.


  • enough of the sequentials for now
  • imaging G2toG4 single color


  • wrote to Maria Ericsson about thicker cryo sectioning (2-3um)

Project 2

  • team meeting
  • see the 3 or 4 posts on this subject from today (protected), and the emails.
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