9:20a – 10:10p,
remotely, 10:50p – 11:30p
- passage cells
- washout probe from new multicolor stain
- finish updating notes from yesterday
- STORM run configurations
- data analysis launched
- break down PRISM2 run of L3C04. (complete)
- break down STORM2 run of L3C03 (complete)
- set up copying and data-fitting of L3C03 and L3C04 data (copied, started fitting C03)
- start making new probes from Lib2: F01, F02, F07, G06, G08, G09
- More probe making
- lower priority, remakes: F06, G05,
- Lib3 on deck: embedded yellow regions E03-E10
- start ChromatinCropper of L3C01
- ChromatinCropper of F05-F06 02/28 data (and 02/27 data?)
- analyze buffer data
New multicolor stains
- New stains failed. I believe I previously verified the low concentration (1.5 uL probe) of these probes makes this happen. Repeat at higher concentration.
Sample C05 on STORM2
- 3 uL COT, 5 uL BME buffer
- staining looks pretty good, spots look decent.
Sample C06 on STORM4
- 5 uL COT, 5 uL BME.
- dots are clearly fainter
- switching pretty well, background is tolerable but a bit higher than desired for imaging small (<50kb) chromatin regions.
- Need to stain new cells today if I want to take adventage of the microscope time I have this week.
- Despite just passaging cells, between the two culture flasks I think I easily have enough cells to plate another 12 coverslips. Let’s fix new cells tomorrow.
- New stains: C07, C08, and double stain F06-A647 + F05-A750.
New probes making
- Lib2: F01, F02, F03, F05, F06, F07, G06, G08
- ran PCR. F05 sublibrary failed, rest look good. Press ahead with all
- ran PCR clean up / DNA clean and concentrate 5.
- setup 20 uL T7 reactions to run overnight.
- all data running last night finished except the 02/28 double color data (which I was excited to analyze). This is still chugging along at a conservative 25% CPU on Cajal.
- canceled and relaunched analysis with 8 parallel processes per color. Along with the C03 analysis this has Cajal up to 100% CPU engagement overnight again.
- email Nicole clarification and explanation of graphs.
- further email discussion and clarifications
- To do:
- normalize all distribution plots. Report both total Ph localizations and total clusters.
- consider truncating distributions at 700 rather than clustering into last bin. It’s important this doesn’t look like a second peak.
- add full length distributions (out to 1.5 um) into supplement.