Tuesday 04/08/14

9:30a – 11:00p

Goals

  • reschedule Dentist
  • try 2, Cajal windows upgrade
  • Need to finish review / referee paper
  • Set up STORM imaging of E3
  • Test new hybes
  • Finish writeup #2 draft for XZ. sent back to team for comments
  • Start writeup #3 for XZ.
  • Meet with Steven to integrate writeup #3

Probe Making

  • qPCR of E5-E12 + F1-F8
  • picked off samples at 2500 – 3000 RPU
  • ran on old gel. Should re-run on fresh gel.
  • Attempting column cleanup using 96-well plates and multi-channel pippettors
    • this is definetely less hands on.
    • and I think per column its cheaper. And spatially smaller
    • spinning at 3000 x g, with open lid columns.
    • TROUBLING: eluted with 12 uL ddH2O, recovered less than 5 uL.
  • setting up in vitro reactions with quick mix.

Cell Staining

  • observed that even without water in the bottom of the box the rubber cement still appears lose and hydrated after O/N stain.
    • Maybe next try keeping lid open / putting slides into oven straight, no box.
    • Maybe skip rubber cement completely? or try a mock without cells, just a coverslip and hybe soln (no probe)?
  • completed hot washes for stains made last night.

STORM

  • imaging E03 sample
  • ran out of time, did not set up new multi-color sample. Will test tomorrow

Cajal Upgrade

  • went through new version of install.
  • configured session-based remote desktop log in first. This didn’t seem to work.
  • configured virtual host based remote desktop. This seems to give the behavior I want.
  • have to add domain users to permissions setting to allow local log in.
  • installed git-scm under Administrator
    • don’t get Git GUI / Git Bash menus in my personal user account.
    • probably should have added desktop icons, can’t find git-gui.
    • maybe just uninstall and reinstall git tomorrow.

Computer Stuff

  • more useful wmic
    • wmic os get FreePhysicalMemory – return the available RAM
    • wmic cpu get loadpercentage – determine total cpu load percentage
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Monday 04/07/14

9:30a – 11:30p

Meetings

  • lab meeting (see protected notes)
  • my journal club presentation (see google-docs presentation)
  • initial planning meeting with Steven — need joint write-up, goal to write by Wed

Writing

  • finalizing write-up 1 for XZ with Jeff.
  • working on rewriting write-up 2 with team.

Chromatin imaging

  • samples incubated an extra 24 hours.
  • no staining! (in either 647 or 750)
  • hypotheses:
    1. maybe the humidity chamber condensation which hydrates the rubber cement dilutes away the probe?
    • try dry hybe comparison tonight
      1. maybe something is really wrong with new hybe dilution mix
    • I think this mix worked sorta the first time the 750 failed last week.
  • took bead images (currently on STORM2 hard-drive, need to copy over).
  • imaging sample E02

New Staining

  • F07-P1 F06-P3 4:4. .33 A647 and .66 A750
  • new E04-P1 2.5 uL to .33uL A647
  • new E05-P1 1.5 uL to .33uL A647
  • use old hybe mix (not hybe dilution mix)
  • stain in box without water (see how this goes)

Fly Work

  • flipped fly stocks
  • balancer Esc almost dead
  • Esc/CyO dead, but Esc/Gla still going strong (if stable?)
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Protected: project 2 notes for XZ

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Protected: Lab Meeting 04/07/14

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hybes, 04/05/14

Chromatin staining,

  • investigating black-yellow interactions

Setup

  • Repeating dual-color hybes with new aliquot of S3 750 labeled secondary. Hopefully this fixes the low specific signal 750 problem.
  • found new hybe dilution buffer not well mixed — dextran sulfate sitting at bottom. Mixed by pipetting to get this suspended.

samples

  • G08-p1, G06-p3 5:5
  • F06-p1 F07-p3 5:5
  • F07-p1 F06-3 5:5
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Installing Windows Server 2012 R2 Standard on Cajal

(not yet validated, need to wipe old hard-drives in a reformat).

Step 1: Configure Local Server.

  1. Select Local Server from the Server Manger Display
  2. Rename the local computer to the name you want to appear in network discovery. You will not be able to change this after domain configuration!
  3. Change time zone if necessary.
  4. Turn on Automatic windows updating. Install updates if necessary.
  5. Deactivate IE Enhanced security for administrators. Otherwise basic web browsing requires 5-50 confirmation clicks every time you want to search or open a new page. This probably only applies to IE, but simply to download Google Chrome from IE with this on took over 100 mouse clicks.
  6. Activate Windows in the server options menu. This will ask for your windows key. You can get this online from the same site where you can download the server iso
  7. install the NVIDIA graphics card

TurnOffIEsecurity

Step 2: Domain Promotion

  • Follows this Guide
  • NOTE: Be sure that AD Tools is installed in the optional features.
    • Otherwise you will not be able to add users.
    • The old add users from control panel approach does not work on domain configurations
    • also be sure the local computer name is set. Windows won’t tell you this in advance, but you can not change it later.
  • You will need to create a security certificate
  • domain set to cajal.local
  • Bio name set to Cajal0 (can’t be same as local name)

Step 3: Install Remote Desktop Software and Add Licenses

  • Follow this Guide
  • In the Add Groups prompt, be sure to Add “Domain Users”. I think it is a good idea to also add “Domain Admins” and “Domain Computers”
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Installing Windows Server 2012 R2 Standard on Cajal – failed attempts

What I did

  • First installed google chrome.
  • Turn off IE enhanced security (one can do nothing on the internet until this done. Just installing chrome required 60 + conformation clicks for all of goggles network checks).
  • install NVIDIA drivers to get graphics card working
  • set up user accounts under control panel. Set up core users as admins

TurnOffIEsecurity

Then I tried to configure remote desktop.

  1. First followed this guide: http://www.wackytechtips.com/installing-and-configuring-remote-desktop-services-rds-on-windows-server-2012/
    • This actually does lead you to a place where you can create enter the remote desktop User/Cal license number
    • this was not sufficient to get remote desktop working
    • I set CAJALusers.cer as my certificate
    • this made references to domain guides
  2. So I tried to add domain users.
    • I followed this step by step guide for setting up a windows 2012 domain controller.
    • https://social.technet.microsoft.com/wiki/contents/articles/12370.step-by-step-guide-for-setting-up-a-windows-server-2012-domain-controller.aspx
    • it wouldn’t let me use Cajal as the domain name (already in use as the local name). So I set it to Cajal0
    • this converted all the users to CAJAL0/username.
    • this still did not enable remote desktop access
    • I think this was a mistake.

I then went through the buttons following the ‘Remote Desktop Services’ on the server manager GUI (which none of the online guides I found mentioned).

RemoteDesktopTool

First, I think this configuration is sufficient to get initial access and you don’t need to do any of the other things. This gets you launched with the 120 day trial at least.

Working through the licenses section I was able to configure things and I think add the licenses that I had built when going through the first GUI. I also created a new security certificate CajalCert.cer

A major bug?

  • after upgrading to a domain controller, I have to log in as domain Cajal0
  • more troubling, I can’t add users. I go to add users, and then it tells me Cajal/newusername doesn’t exist, and that Cajal0/newusername doesn’t exist. I know it doesn’t exist, I’m trying to create it!

I think the domain controller addition was a mistake, so I set out to downgrade it following this guide: http://terrytlslau.tls1.cc/2012/03/domain-controller-demotion-on-windows.html

Was wrong about domain user

  • Turns out this is necessary, remote desktop services button doesn’t display anything

Start again from scratch

Basic configuration

  1. Deactivate IE secutiry
  2. Activate Windows in the server options

Active Directory Domain Services Configuration

  • domain set to cajal.local
  • Bio name selected Cajal
  • Server name is stil WIN-KF8BIF09SKR

Third time’s the charm

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Thursday 04/03/14

10:00a – 1:00a

Goals

  • Data for Jeff
    1. Density distributions for over sequence for template and probe from my library
    2. Cumulative dist plots for probe abundances from my library
    3. Insertion/deletion/mutation error plots as a function of bp for template and probe
  • New figure for Ting
    1. Quantification of signal per spot as a function of labeling scheme, with images of each.
    2. Quantification of background per cell as a function of labeling scheme, with images of each.
    3. Quantification of on-target to off-target probe binding frequency.
  • Upgrade Cajal to Windows Server 2012
    • upgrade took all day and failed
    • I think I learned a lot about how Windows Server 2012 has changed.
    • see posts on subject.
  • STORM4 imaging
    • serious issues with camera alignment
    • serious issues with quadview alignment
    • probably issues with back-beam alignment secondary to camera misalignment, can’t diagnose.
    • laser connectivity issues to GUI?! GUI stopped talking to 642 and 561 laser. not good not seen this before.
    • unable to resolve all issues in time for an overnight run.
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Tuesday 04/01/14

10:30a – 1:20a

STORM imaging

  • Microscope computer blue-screen crash during the night on prism2
  • Jiang reports having seen similar issues on prism2

Presentation Prep

  • Making slides for Friday’s team meeting with Wu group
  • see googledocs presentation. This took the half of the day not spent on the PH project.

Ph project

  • see discussion log
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Protected: Ph Polymerization: on modeling

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