9:30 am – 5:15 pm, 8:20 pm – 11:30 pm
Ph-polymerization
Manuscript to do
- STORM section, reply to notes in main text
- Finish Layout Supp figure 2 and Supp figure 4
- added supplement captions form both
- Check the legend to Figure 5 (new model) and sup. Fig. 11 (rest of model fig)
- Compile everything into a PDF
- Check response to reviewers.
STORM
- imaging on STORM2
- 2 color
- MEA COT buffer
- sample quality (ANTC-750 + G6-647)
- still an odd background nuclear glow in 647 channel (not related to STORM-dye switching, uniform, unbleaching. Still suspect some contaminate Draq5 or something of the sort. Maybe the formamide overnight treatmetn helped drop this down, the STORM switching on top of this background looks pretty good.
- 561 beads just switch dark / bleach quickly in the MEA COT buffer
- swapped fresh buffer, added fresh beads. Conventional imaging beads nice and bright
- start STORM imaging, and beads go dark pretty quick and don’t come back
- I recall this happening before, and only lasting the first few movies
- indeed the beads do come back bright, but now the 750 switching looks bad.
- probably the buffer quality is decreasing, favoring the beads and disfavoring the dyes again. Unfortunately the switching quality in 750 is a no-go at this point.
- I want to try this again in BME
- switched buffer to BME.
- beads don’t recover very well. They aren’t as completely dim but its still pretty bad. Should replace with fresh beads again.
- 750 switching not as bright, and most dramatically, not as switchy — fewer blinking dyes for way less long. Need to recalibrate all my ramp files
- using substantial 405 I can get some switching. Let’s see how this goes
- next I’ll try MEA alone, no COT.
- switched to MEA no COT
- first movie, 750 dim but blinking, 45K frames instead of 70 (better than the BME, but we’ll see if it lasts)
- at least the newly added beads stay bright
- running the MEA overnight.
- still a total unusual amount of background in the 647 channel
Cell culture
- Psc null cells growing well. I have 3 vials in freezer so hopefully those are fine if I need them again.
- Terminated Psc null flasks
- trimmed down to 1 KC in SFX flask and 1 KC in Schneider’s flask.
- interestingly the Schneider’s cells are now adhering better than the SFX
Seq Prep
- Tape station training
- tape needs to be reserved online
- ordered 1 tape for next set of experiments
- HS DNA is the recommended tape for library prep.
- protocols sent in email.