10:00 am – 1:10 am
Discussion
- sent feedback to Ting about proposed revisions for fig2
- sent comments to Hao about presentation for this evening
- project 2 team meeting
- meeting with XZ discuss project 2 partitioning (see emails).
Analyzing STORM data
- C10 Analyzed through image 23 (50 spots)
- C11 Analyzed through image 29 (47 spots)
Sequential Staining
- imaging new double stained slide, first stain: BX-C-P3 (looks absolutely beautiful, clear high contrast staining).
- kept UV power below 10%. imaged 110K frames.
- should add these to the data collection even if the F06 doesn’t pan out
- added P3-rc toehold probe (more like leg-hold, with 20 bp homology) to solution. Can’t see spots in hybe solution
- all extremely bright spots completely gone in 30 min.
- rehybe with S1 secondary + P3-rc probe to target F06-P1. 1 hr + wash.
- re-add STORM buffer. took some conv and started STORM movie of new test spots. Some substantial background + spots not very bright (as in scarcely detectable on conventional). Wash more, than add more STORM buffer.
- spots still faint. took some conv and STORM movies
- Observation: P3-rc and S1 have substantial complementary structure (10 bp) because we use 10 bp of the common in the S1 binding site. This is not true between P3 and S2 or S4.
- also since all the toehold probes have
- rehybe overnight at 37C heated objective with S1 alone
To try next
- with BXC+ Fo6,
- switch order of probes stained. F06-P3 + BXC-P1
- use just the secondary competetor strand, not the toehold version.
Hybe Prep:
- new hybe dil buffer (1.25x)
- 12 mL dextran sulfate
- 6 mL 20X SSC
- 0.6 mL 10% Tween-20
- 30 mL formamide
- 1.4 ddH2O
Mentoring
- helping Bogdan got sorted with buffers