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
- NO STAINING!
- 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.
STORM
- enough of the sequentials for now
- imaging G2toG4 single color
Communication
- 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.