Alistair's Lab Notebook

9:15 am – 9:15 pm

Ph project

• discussion clarifying “cluster” definition — yes this goes all the way down to “clusters” which probably contain only a single molecule.
• analyzing PH clusters lacking PC
  • this is not as robust with the 750 data as with the 647 data
  • need some adjustments for background and density

Project 2

• team meeting
• data analysis — see post.

Chromatin imaging

• need new z-calibration movie
• modify CC to use bk images for mask and original scan images for conv. Ideally have toggle to use either set in either place.
  • currently 2 channel nature of the data is not detected if we use single channel conventional images.
• fixing some issues with chromatic alignment — turns out I entered the channels in the wrong order in STORMrender, and then got myself confused. CC and STORMrender agree perfectly, CC for the 750 data takes the guesswork out.

Chromatin data analysis

• analyzing F06 F07 data
• 750 background pretty high. Not certain the z-focus difference is perfectly corrected
• new 750 z-calibration seems to be needed.
• despite skipping lots of spots for background issues or low localization number, I can still get 50 cells in this data set.

9:30 am – 10:00 pm

STORM

• L2 F02 F03 — F03-p3-750 not very bright given size
• F02 stain looks like it could be better — a bit of background here too
• imaging L2 F02 F03 anyway, shorter exposure
• 9 pm set up imaging of L3 E08-p2-6 E09-p3-7
• not as bright as expected for these loci. Remade Glox buffer (6 days old now), spots brighter

Probe Making

• ChrLib4 probe synthesis A01-B12

IVT reactions

• 30 uL total :
• 10 uL buffer nucleotide mix
• 10 uL DNA template
• 1 uL RNAsin Plus
• 1 uL T7
• 8 uL ddH20 nuclease free

25 x master mix

• 250 uL buffer nucleotide mix
• 25 uL RNAsin Plus
• 25 uL T7
• 200 uL ddH20 nuclease free

RT

• 3 uL primer
• 2 uL dNTP
• 14 uL RNA
• 6 uL RT buffer
• 1 uL RNasin
• .5 uL Maxima
• 4 uL ddH2O

25x RT P2

• 75 uL primer P2
• 50 uL dNTP
• 150 uL RT buffer
• 25 uL RNasin
• 12 uL Maxima
• 100 uL ddH2O

6x RT P3

• 18 uL primer P3
• 12 uL dNTP
• 36 uL RT buffer
• 6 uL RNasin
• 3 uL Maxima
• 24 uL ddH2o
• tubes 1,3,8,17,19

cleanup

• froze samples, try plate-column cleanup tomorrow.

PH polymerization

• working on manuscript revisions / additional data analysis.
• quantify total overlap by fraction of localizations of one channel within localization precision (~25 nm) of other channel. I had done this for PH-WT and not PH-ML before. Quick estimate drawn from last cell of PH-ML data (20% PH-ML with PC and 30% PC with PH-ML). Should go back and compute all cells and average.
• quantifying distribution of non-co-clustered populations — this we basically had already, just plot the ~coclustered mlists and run cluster size stats on those.
• quantifying correlation of cluster sizes. — finished off analysis started yesterday. Approach uses an OR mask and then overlap to call clusters, so all localizations from two clusters that share a portion of pixels are counted — this reduces artificat from partial misalignment on the 20-50 nm scale due to residual uncorrected drift and residual uncorrected chromatic aberration.

Other Notes

• new GSA membership

7:30 am – 5:15 pm, 10:15 pm – 12:20 am

Chromatin STORM

• setting up imaging of L2 F01-A750 F02-A647
• a bit dim but clearly detectable

Project 2

• working on data analysis with Steven’s new data.
• see protected post.

(volleyball – 11:00am-12:30pm)

PH project

Manuscript

• new manuscript draft from Bob/Nicole/Ajaz
• some additional modifications to the figures coming soon.

Data analysis

• look at size distribution of non-overlapping clusters.
• analyzing size correlation in PH-PC clusters

10:00 am – 9:00 pm

STORM

L2 F06F07

• finish hot-washes
• add beads, make STORM buffer
• Imaging F06-p3-7 F07-p2-6 double color
• staining in both channels bright and clear
• colocalization looks decent

New buffer for multicolor STORM

• 2.5 uL MEA
• 1.5 uL BME
• 5 uL COT
• 10 uL GLOX

Writing

• writing up formal comments on manuscript for colleagues
• # need to write abstract of DR retreat!

New Probe making

regions

• internal black domains: A01-A10 + E01-E10
• some BXC small pieces: A11-B12 + E11-F12
• 24 new probe regions in all (plus let’s do some in 2 color).

per well

• 25 uL 2x Phusion master hot start
• 2.5 uL Eva Green
• 5 uL library
• 12.5 uL ddH2O
• 2.5 uL Fwd Primer
• 2.5 uL Rev Primer

27 x master mix

• 675 uL Phusion hot start master mix
• 67.5 uL Eva Green
• 135 uL library
• 337.5 uL ddH2O

Cleanup

• ran cleanup using Zymo 96 well plate.
• much easier than individual spin columns

7:30 am – 10:00 pm

(9:30 am – 11:30 am volleyball)

RT reactions

Probes in pipeline

• L2 F01 – F07
• L3 E06 – E09
• making with both P2 and P3

Per RT

• 15 uL RNA
• 4 uL primer
• 6 uL RT buffer
• 2 uL dNTPs
• .5 uL RNAsin
• 2 uL ddH2O
• 0.5 uL Maxima
• Total volume = 30 uL reaction

12 reactions master

• 48 uL primer
• 72 uL RT buffer
• 24 dNTPs
• 6 uL RNasin
• 6 uL Maxima
• 12 uL ddH2O
• now OUT of Maxima
• now OUT of RNasin

digestion and cleanup

• added 2x volume of EDTA + NaOH (equal volume would have been sufficient)
• volume for cleanup now greater than 1 column
• accidently starting adding on P2 probes E6 through E9 in backwards order (should have kept this in ordinal order at the T7 stage).
• Lib

Test gels

• all my precast gels have been used
• started casting new gels
• acrylamide won’t polymerize today
• added more TMED and APS, still no polymerization

Cell staining

• new stains:
• F06-p2-A647 + F07-p3-A750
• F07-p2-A647 + F06-p3-A50