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

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 CajalDomain.local
  • NETBOIS name set to Cajal0 (can’t be same as local name)

Step 3: Install Remote Desktop Software and Add Licenses

  • Follow this Guide
  • self signed certiicate name: Cajal.CajalDomain.local
  • store the certificate: Users\Aministrator\Documents\CAJAL.cer
  • 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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Monday 03/31/14

9:45a – 12:25a

Chromatin Project

Microscopy sessions!

STORM2 Lib2 mutlicolor imaging.

  • make fresh MEA
  • make fresh Glox
  • check stains, find a good stain to image Wed or Thur night.
  • if cells and spots are dense 10 regions can be a good start.
  • substantial Microscope Alignment issues
    • using an iris appeture in place of the objective and the spot on the ceiling, it is clear that the main column is way out of line.
    • the camera is well off of line relative to the main objective, because after aligning the iris and the roof spot so we have a clean aligned column coming up from the dichroic mirror through the objective, the field looks pretty flat but is cut almost in half by the apetures in the quadview.
  • The TIRF controller is crazy on this setup and needs to be replaced – it starts moving of its own accord at random.
  • imaging sample F7-6 F6-7
    • spots very bright and strong in 647 channel
    • cells very sparse on this coverslip 🙁
    • 750 spots hardly detectable in conventional image, very high background. I swear I have gotten much much brighter 750 spots.
    • STORM of 750, do see spots, just also have lots of background. Tons of switching, sometimes bright. On average way less bright than I was getting earlier with this MEA COT .5:1x buffer mix. * Not sure what that’s about. Maybe this COT aliquot is dead.
    • need to compare G2G3 control
    • G2G3 control conventional images still pretty faint, readily detectable but not too strong. Not like previous 750 images.
  • For comparison, here’s a 750 conventional selected at random from the 2/15 dataset. Not sure if that day was particularly good anyway, but the spots are clearly brighter (max = 1000 in this image).

A750_spots_140215

STORM3 imaging with Pallav

STORM1 single-color imaging Lib3 E01

  • checked alignment of back-optic, looks reasonable. Adjusted somewhat to center on roof spot. (should have cranked the pinhole before the defocusing mirror down to shrink the beam spot for greater sensitivity).
  • checked alignment of dual-view in bypass mode to camera, also looks reasonable.
  • Conventional images look great
  • STORM not as bright or switching as fast as desired.
  • Lots of swtiching, easily 200K frames. molecules not as sprightly as desired. spotfitter seems to make these look smaller than they look on the screen based on the frequency of overlapping dots. This sample is also definetely one for DaoSTORM. Overlapping double activations quite frequent.
  • Potential issue with EO1 identity — locus region and name don’t match properly in library table online. need to check this.
  • Position 3 or so loses focus in during scan. This O/N is probably not going to work too well :(.
  • Maybe I should try STORM 3 for more laser power and just use the blead-through beads (I think I can get these to show up just fine).

STORM4

  • continue running project 2 experiments and controls
  • see notes

Commitments

  • Accepted paper to Review. Due in 10 days.

Fly work

  • Flip fly stocks! (postponed today).
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Protected: project 2 experiments 03/31/14

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