Protected: Highlights for lunch meeting

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Tuesday 07/22/14

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

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Monday 07/21/14

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

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Protected: lab meeting 07/21/14

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Protected: project 2: E2

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Wednesday 07/16/14

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

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Protected: project 2: E2 analysis

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