Tuesday 07/29/14

9:00 am – 5:30 pm, 9:00 pm – 12:30 am

Goals

  • discussion with Chong about project
  • washout probes from new hybes
  • break down STORM run, transfer and fit data
  • spot brightness analysis
  • flip fly stocks (?) — not managed

Chromatin project

New library design

  • designing new probes for En and B09 (yellow ~100 kb) that alternate probes with different primers
  • will synthesize these with P2 and P3 primaries.

Sequential staining control

  • imaging BX-C-p3-A647

New Stains

  • E08-p1-A647 + E09-p3-A750
  • F06-p1-A647 + F07-p3-A750
  • F07-p1-A647 + F06-p3-A750

Project 2

  • see summary
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Protected: project 2: E8 analysis

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Protected: project 2 Photon quantification 07/29/14

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commentary on the 1%

I just read this article highlight in Nature about the distribution of publication output that is so misleading I feel compelled to write about it. The finding of the original article by Ioannidis is that only 1% of researchers publishing between 1996 and 2011 published every year. The implication/spin given to this finding is that the top 1% of scientists are contributing disproportionately (41%) of the scientific output — a ludicrous misinterpretation. The fraction is so low simply because most people who publish happen to be graduate students and post-docs (just pick any scientific paper and go through the author affiliations) and very few of those are in the game for 15 years. For example, lots of the authors in the denominator in this list were graduate students on papers published between 2000 and 2011, very few of whom were full time scientists in 1996 and thus immediately eliminated from the publish-every-year numerator. This 15 year time period is long enough to cover plenty of productive, publish every year grad students who became publish every year post docs who became publish every year junior PIs between 1997 and 2011 and still don’t make it into the numerator which requires they published in 1996 before their scientific career began.

At the very least the denomenator could have been out of researchers that started publishing in 1996, what fraction continued to publish every year there-after. This of course would still be rather unuseful statistic, since the amount of hours required to lead a project (typically denoted by first authorship, also typically carried out by a graduate student or post-doc) is vastly in excess of the amount of hours required to be senior author. Not to diminish the critical role of senior authors in funding, often designing, and usually mentoring a project. I doubt most PIs with a lab of N people believe they contribute N times more to science than their average group member, though if all group members include the PI on their publications the PIs publication rate is N times higher.

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Monday 07/28/14

10:00 am – 5:20 pm, 10:00 pm – 11:50 pm

Goals

  • prep new cells for multicolor hybe
  • continue analysis of E7 data

Lab meeting

  • Ruobo presentation – see protected notes
  • Jiang journal club — axon mylination in response to neural signaling

Project 2 analysis

Cell prep

  • prepping cells fixed yesterday for hybridization
  • in RNase treatment in warmroom (Hao is using new hybe oven and probably best to keep that RNase free)
  • now in 50% formamide at 4C ready to hybe.

new stains

  • 2 large coverslips of BXC P1 + P3 for double labeling control against movement
  • 1 large coverslip of L3Eo6toE09 for double labeling control against movement

STORM

  • imaging L3E09 PH in S2 cells
  • 647 switching looks much better than when imaged with BX-C. Maybe should repeat BX-C
  • 750 staining looks great but deactivates quickly
  • might try more imaging of this slide with 750 STORM. should probably do in MEA instead of BME
  • use mosaic tile to get conventional images if STORM is already inactivated (just match stage location?)
  • need more COT
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Protected: lab meeting 07/28/14

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Protected: E7 summary

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Protected: More E7 error analysis

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Friday 07/25/14

10:00 am – 10:00 pm

Prep for XZ meeting

  • see slides
  • conclusions from meeting
    • more dual color imaging
    • need dual labeling of same sample as a control. Try mixed BXC labeling (F03-F05 in p3 and p1 at matched concentrations and just hope we get enough of both).
    • also should design for next library the alternating probe approach to do this more carefully, in case the mixed probe approach doesn’t work.
    • discuss schedule with Bogdan for black regions

Further analysis of sequential data sets

  • 2014-07-02_L2-BXC-F06 — 2nd channel / hybe no decent data
  • 2014-07-04_L2-BXC-F06 — 2nd channel / hybe no decent data
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Thursday 07/24/14

9:45 am – 11:00 pm

Goals

  • XZ meeting prep
    • updated plots of y-internal and b-internal
    • multicolor update
    • low photons
    • sequential method I
    • sequential shadows
    • toe hold probes
  • Sequential staining
    • test S3 first stain
    • test wash out with toehold S3toe5
    • test restain with S1
  • Fig prep / data analysis
    • crop y-internal regions for fig
  • comments on manuscript (see email)

Sequential Staining

  • heating objective
  • heating wash buffer
  • adhere beads to sample
  • make new STORM buffer
  • Imaged ~8 regions of E09-P3
  • Piezo not on — some notable drift may have happened :(
  • attempting to remove S3 probe with S3toe5 at 2:1000 hybe dil buffer for 30 min at 37C (imaged cells during incubation)
  • most of the decrease in signal appears to be due to bleaching, judging by near-by non-imaged regions.
  • attempting to remove S3 probe with S3toe5 at 10:1000 in hybe dil buffer for 30 min at 37C
  • second treatment at 1:100 dilution works! Took some images.
  • washout with 50% formamide 2x SSCT at 37C ~10min
  • hybe S1 at 2:1000 hybe dil buffer at 37C
  • washout stain, 20 min
  • add new STORM buffer and image. Dots are nice and bright
  • Dots are perfectly clear in previously imaged regions, but a bit dimmer. I think the high (330 mW 647 laser) may not be ideal.
  • spots not imaged don’t actually STORM that much better (imaged after rnd 2 imaging completed).
  • maybe just a longer hybe and higher secondary concentration would be better?

Analysis of Sequential Stain data

  • L2F03F04 from 2014-05-29
    • hybe2 mostly failed (due to laser damage?)
    • some images have weak staining

F03F04seqHybe hybe2laserDamage

PH-project

  • treating 1 PH-M 1 PH-Wt and 2 S2 coverslips
  • 30 min in 5% FA + 1/2x PBT for post fix
  • PBS wash
  • 30 min in PBS + RNase A
  • wash in 2X SSCT
  • prehybe in 50% formamide + 2X SSCT
  • stain PH-M, PH-Wt, and S2 with 750-BXC
  • stain second S2 with 750 L3E09

Data analysis

  • Merging old and new BXC data
  • be careful, need to change source-folder for internal scaling data for BX-C back to old
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