Wednesday 07/23/14

Posted on July 23, 2014 by admin

10:00 am – 12:15 am

Chromatin Project

Sequential staining

  • snapped coverslip
  • tried mounting half on flow-system anyway
  • this leaks — not enough pressure seal in the gaskets (especially relative to pump pressure of manual syringe)
  • try manual hybes of second broken piece of coverglass
    • hybe on glass slide at 47C 30 min
    • wash in 50% formamide 2x SSCT at 37C 20 min
    • This failed: coverslip upside down 🙁
  • try manual hybes with failed stain of the mounted (larger) half of the broken coverglass
    • hybe on glass slide at 47C 30 min
    • wash in 50% formamide 2x SSCT at 37C 20 min
    • Still failed: very weak spots, not all cells have evident spots
    • either 30 min is not enough time to hybe or this probe failed (which could explain why the flip failed).

Summary of S1 S3 toehold replacements

SequentialStainIssues

New Stains

  • Goal: more troubleshooting / testing of sequential staining
  • L3E08-p1 + L3E09-p3 + S3-A647
  • L3E09-p1 + L3E08-p3
  • L3E08-p1 + L3E09-p3
  • L3E09-p1 + L3E08-p3
  • 4 ul of each probe diluted into 120 uL hybe dilution buffer, split 2 ways (50-60 uL each)

Chromatin Data Analysis

  • ChromatinCropper Analysis today
    • Finished ChromatinCropper analyzing BXC (last 2 cells)
    • ChromatinCropper of F03F04 data — these spots are much smaller than I thought from the quick fit data.
    • E06 (repeated for lower background)
  • renamed new BXC_data.mat file as BXC2_data.mat and moved to 2014-07-09_BlueInternalData
  • Analyzing G2toG4 data. Looks pretty good. Similar size as ANTC
  • launched analysis of E07toE09 data and E07 (rpt 3) data.
  • G05 automatic length extraction from name is incorrect — 40 bp, should be 64 !!
  • F4, G2, G3, G4 should be double-checked

XZ meeting prep

  • Put in draft fig 2.
  • bead data for G2toG4 not analyzed, processing now

Simulations

  • RC now added openMM to python paths. should try again to see if we can get this to run at all.
  • no longer gives error on import simtk.openmm.openmm
  • now errors on import dumpOutput

Project 2

  • team meeting
  • work on code
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Protected: Highlights for lunch meeting

Posted on July 23, 2014 by admin

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Tuesday 07/22/14

Posted on July 22, 2014 by admin

11:25 am — 11:45 pm

(biking Minuteman path, 8:15a-11:25a)

Goals

  • Ajaz’s sample
  • project 2 data analysis
  • chromatin data analysis

Project 2 analysis

  • see protected post

Chromatin Project

Data analysis

  • fitting E06 and G2toG4 data
  • trying to analyze BX-C data from last run of BX-C F06. Drive communication extremely slow.
    • aborted data transfer from STORM2, speeds up disk communication
    • these spots look great (as anticipated).

New probes

  • L3 E06 to E09 (4)
  • L2 F01 to F07 (7)
  • Per well
    • 25 uL Phusion
    • 15 uL ddH2O
    • 3 uL 1:50 library
    • 2.5 uL EvaGreen
    • 2.5 uL Fwd primer
    • 2.5 uL Rev primer
  • 8x Master Mix Lib2
    • 200 uL Phusion
    • 120 uL ddH2O
    • 24 uL Lib 2
    • 20 uL EvaGreen
  • 5x Master Mix Lib 3 1:50
    • 125 uL Phusion
    • 75 uL ddH2O
    • 15 uL Lib 2 1:50
    • 12.5 uL EvaGreen
  • To do: BXC-pieces
    • A11 to C08 (fwd primers)
    • E11 to G08 (rev primers)

New stains?

  • need to fix more cells
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Protected: project 2: 07/22/14

Posted on July 22, 2014 by admin

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Monday 07/21/14

Posted on July 21, 2014 by admin

9:30 am – 5:00 pm, 9:30 pm – 11:45 pm

Lab meeting

  • 10:00am – 1:30pm
  • Steven presenting, see notes

Project 2 analysis

  • see protected post

STORM

  • imaging L3 E07toE09
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Protected: project 2 update

Posted on July 21, 2014 by admin

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Protected: lab meeting 07/21/14

Posted on July 21, 2014 by admin

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Protected: project 2: E2

Posted on July 17, 2014 by admin

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Wednesday 07/16/14

Posted on July 16, 2014 by admin

10:00 am – 12:00 am

Sequential Staining

  • all at 37C (heated objective)
  • flow 2X SSCT to rinse out storm buffer
  • prehybe in 2x SSCT + 50% formamide, 15 min
  • hybe 30 min with S1toe5 (3uL in 1 mL hybe dilution buffer)
  • wash 15 min in 2x SSCT + 50% formamide
  • rinse in 2X SSCT
  • flow STORM buffer and image sample — all staining removed. So toe 5’s work.
  • rinse in 2X SSCT
  • prehybe in 2x SSCT + 50% formamide
  • flow hybe S3 (3uL in 1 mL hybe dilution buffer)
  • get DC power supply for heated chamber, heat to 37C (reading 28C at surface sensor).
  • hybe S3 3 hour.
  • wash out hybe 3
  • NO STAINING!
  • apparently 15 bp is too much to remove (Still don’t understand why the toehold doesn’t work in reverse here, though I guess this isn’t really a toehold situation, its a ‘slap away’ the invading strand — we have a 15 bp anchored duplex with a 30 bp tail guarding a non-homologous 30 bp of sequence that the secondary wants to bind.

STORM

  • enough of the sequentials for now
  • imaging G2toG4 single color

Communication

  • wrote to Maria Ericsson about thicker cryo sectioning (2-3um)

Project 2

  • team meeting
  • see the 3 or 4 posts on this subject from today (protected), and the emails.
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Protected: project 2: control

Posted on July 16, 2014 by admin

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