Alistair's Lab Notebook

9:30a – 3:45p, 7:00p – 9:00p

schedule

• 11:00a-12:00p, volleball match
• 1:45-3:00p doctor’s appt
• 3:45p-7:00p feeling sick gone home

Chromatin

STORM

• imaging B09

Data analysis

• Analyzing small region yellow data: B03 and B04

10:00am – 7:00pm

Goals

• Call Otto’s order pizza for lab meeting (done)
• Finish lcPCR and cleanup (done)
• Set up T7 reactions (done)
• post-fix Ajaz samples to try immuno-FISH again (done)
• stain cells (done)
• finish letter and reply to Nicole about collaboration (done)
• review and send fellowship progress report to XZ (done)
• Review manuscript draft for Max (post-poned)

Making probes:

probes

• B10-B12 (last of the yellows)
• C09-C12 (last blue, first 2 blacks)
• D01-D07 (all the end-to-end blacks)
• [D08-D12 are Bogdan’s internal blacks]

Progress

• Ran QPCRs
• Ran PCR cleanup
• running 30 uL T7 reactions O/N

Cell Staining

• post-fix in 1% GA 4% PFA 1 hr.
  • 1. S2 anti-PH
  • 1. PHwt anti-flag
  • 1. PHmut anti-flag
• washed with PBS, then 2x SSC, then prehybe.
• Stain BXC and ANTC
  • G02-P2-cy3
  • F05-P1-405 (old sample) + S1-488

New Probes to stain

• E11 + E12 + F01 (G06/alphaTub locus)
• E11
• E12
• F01 (alpha Tub itself)
• B08
• B09
• all with P1-405 + S1-A647

Ph Project

• remade figure for neg controls (separated channels, matched cropping boxes)
• sent figures for neg controls to Ajaz
• review figures
• finish draft letter for Nicole and sent back

10:30a – 4:00p

New probes to make:

• B10-B12 (last of the yellows)
• C09-C12 (last blue, first 2 blacks)
• D01-D07 (all the end-to-end blacks)
• [D08-D12 are Bogdan’s internal blacks]

master mix

• 525 polymerase (25*21)
• 420 ddH2O
• 21 of Lib3 (1:50 dilution)
• 45 uL to each well
• 2.5 uL of F-primer,
• 2.5 uL R-primer

9:30a – 7:00p

Goals

• Finish progress report
• limited-cycle RT PCR for new probes
• enter into database last batch of ~30 probes synthesized
• Analyze new 2-color data

Literature

• interesting article by Fontana on aging driven by random-gene expression when lacking strong selective pressures operative during earlier development
• interesting discussion in Science on inequality

Analyses to do

• re-bin Kc Hi-C data
• radius of gyration detailed analysis

Coding / library design

• installed java on Cajal and added to system path.
• installed oligoArrayAux. Needs to go in Program Files, not Program Files x86 to work with oligoArray.
• now working on Cajal. currently launching at 3 sec per gen, and limted by launch rate (CPU rarely kicking over 40% and that’s with dotfinding running in the background).

11:00a – 12:45a

(9:00a-11:00a, volleyball practice).

Meeting with Bogdan

• see notes
• Bogdan Data: \cajal\AlistairTemp3

Chromatin Data Analysis

Chromatin Cropper Coding to do

• integrate radius of gyration computation
• integrate flip h/v/transpose options for conventional images
• fix auto-loading of conventional images (currently not loading 000n conventional images correctly, instead loads first image from movie stack).

Troubleshooting

• implemented flip h/v/transpose (FlipImage, now in ImageTransforms in matlab-functions)
• this is not actually the problem, it is just the name matching that is the issue.
• missing some conventional images for the L3 F03-p1, F02-p3. Just find spots from STORM images (the contrast is good enough for these).

Analyzed data

• saved data in C:\Users\Alistair\Documents\Research\Projects\Chromatin\Data\2014-04-23_Lib3data2clr

Data that needs attention

• E02 data staining a little weak (and many section in bad focus).

Sequential Staining and Imaging

• set up objective heater and bio-optics chamber
• objective heater movable collar broke (seems to have happened after inappropriately loosening the screws on the side instead of the back of the unit
• black spacer broke off. Epoxied this back on.
• removed tiny hex-screws holding down top to expose mechanism. Plugged in loose side of collar and resealed with set-screw. Device now fixed!
• heating objective (should have started this earlier, takes 1 hr+).
• 5:45pm, started incubation in secondary. 1:100 dilution of 100 mM stock into 50% formamide 2x SSCT, diluted a further 1:4 into hybe dilution buffer.
• Imaging sample Lib2, F03-p1, F04-p3
• cannot find S3-A647 secondary (supposedly ordered on 12/18/13). Also cannot find P2-A405 supposedly ordered at same time.
• proceed with S3-A750, just do sequential multi-color :(.
• ordered new primers, 5′ A405 for P2 and P4, 3′ A647 for S2, S3, and S4.
• both sequential hybridizations look excellent.

10:00a – 11:00p

STORM

• imaging F04F03 samples
• finish staining / washing B05 + new F03-F04 no secondary, F05-F06 no secondary (see labels on wells)

cell staining

• Prep 12 new 18mm coverslips for staining
• Prep 3 new 40 mm coverslips of cells.
• 1 of 3 40 mm coverslips all cells detached after LN2 step.
• stain F03-p1 + F04-p3 (check this), no secondaries, on 40 mm coverslips.
• stain F05-p1 + F06-p3 (check this), no secondaries.
• stain B06 and B07 (probe prepped yesterday).
• 40mm coverslips staining 40uL hybe mix + 3-4uL probe, inverted on a regular coverslip and sealed just on 2 edges.

Writing

• working on Progress Report for Damon Runyon (see googledocs)

project 2

• see notes