User:Steven J. Koch/MTC/April 7 2011: Difference between revisions

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#* Observations, make figure(s)
#* Observations, make figure(s)
#* Upload at least one to figshare.com (requires linking to your Science 3.0 account)
#* Upload at least one to figshare.com (requires linking to your Science 3.0 account)
==Microtubule data==
Below you will find the data that we took in the ''lab tour'' class. Both movies have the following characteristics.
* We took data at 5 frames/s for a total of 10 minutes.
* The movies have been sped up by a factor of 6 so you are watching it at 30 frames/s.
* The movies are false colored using ImageJ's ''Green Fire Blue'' LUT.
* Fluorescent images were obtained with rhodamine tagged tubulin. The tubulin was sourced from Cytoskeleton and was polymerized into microtubules using a 29:71 ratio of labeled tubulin:unlabeled tubulin. They have been fixed with 10 μM Taxol.
* The EMCCD gain of the camera was 150.
* We used a 100 W Hg lamp attenuated by 94%.
* The objective used was a 1.42 NA PlanApo 60x objective held at a constant temperature of 33°C.
* The pixel length is 166.7 nm/pixel which gives the field of view dimensions of 82x110 μm.
* The glass was passivated with 1.0 mg/mL bovine alpha casein.
* The concentration of kinesin used was 27.5 μg/mL.
For a complete description of how the assay was prepared and run, please see [http://www.openwetware.org/wiki/User:Andy_Maloney/Kinesin_%26_Microtubule_Page this page that describes a basic procedure on how to conduct a gliding motility assay].
<font size=4>'''Note:'''</font> To complete this assignment, you must make comments about the movies. Be sure to add your signature to the comment by including <nowiki>'''~~~~:'''</nowiki> before you comment. Write down any and all observations you can make about the microtubules. Try not to overlap comments with your fellow students but, if you do, be sure to make them such that a discussion is started. Feel free to leave questions for Dr. Koch in the discussions below.
===Microtubules polymerized in D<sub>2</sub>O===
<html>
<object width="480" height="390"><param name="movie" value="http://www.youtube.com/v/wOQT9X0mirA?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/wOQT9X0mirA?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="390"></embed></object>
</html>
====Add comments below====
<!-- Add your comments below this line -->
===Microtubules polymerized in H<sub>2</sub>O===
<html><object width="480" height="390"><param name="movie" value="http://www.youtube.com/v/uEc3HmGloNo?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/uEc3HmGloNo?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="390"></embed></object>
</html>
====Add comments below====
<!-- Add your comments below this line -->

Revision as of 08:03, 15 April 2011

MTC April 7 2011

Molecular Motors

Microtubules field trip / HW--With Andy Maloney

  1. Read up on Tubulin / microtubules
  2. Design Experiment
    • "Tweak" the tubulin polymerization solution--Andy will prepare it. Don't do same as someone else.
      • A boring example: dilute the tubulin, expecting fewer or longer MTs.
      • Try not to do something "crazy."
      • We can't change temperature (at least not easily)...polymerization time is an option
    • Due Tuesday April 12 -- you can email it to me.
  3. Perform Experiment
    • Thursday, April 14 9:30 AM At the CHTM, Don't Be Late!
  4. Analyze data
    • Observations, make figure(s)
    • Upload at least one to figshare.com (requires linking to your Science 3.0 account)

Microtubule data

Below you will find the data that we took in the lab tour class. Both movies have the following characteristics.

  • We took data at 5 frames/s for a total of 10 minutes.
  • The movies have been sped up by a factor of 6 so you are watching it at 30 frames/s.
  • The movies are false colored using ImageJ's Green Fire Blue LUT.
  • Fluorescent images were obtained with rhodamine tagged tubulin. The tubulin was sourced from Cytoskeleton and was polymerized into microtubules using a 29:71 ratio of labeled tubulin:unlabeled tubulin. They have been fixed with 10 μM Taxol.
  • The EMCCD gain of the camera was 150.
  • We used a 100 W Hg lamp attenuated by 94%.
  • The objective used was a 1.42 NA PlanApo 60x objective held at a constant temperature of 33°C.
  • The pixel length is 166.7 nm/pixel which gives the field of view dimensions of 82x110 μm.
  • The glass was passivated with 1.0 mg/mL bovine alpha casein.
  • The concentration of kinesin used was 27.5 μg/mL.

For a complete description of how the assay was prepared and run, please see this page that describes a basic procedure on how to conduct a gliding motility assay.

Note: To complete this assignment, you must make comments about the movies. Be sure to add your signature to the comment by including '''~~~~:''' before you comment. Write down any and all observations you can make about the microtubules. Try not to overlap comments with your fellow students but, if you do, be sure to make them such that a discussion is started. Feel free to leave questions for Dr. Koch in the discussions below.

Microtubules polymerized in D2O

<html> <object width="480" height="390"><param name="movie" value="http://www.youtube.com/v/wOQT9X0mirA?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/wOQT9X0mirA?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="390"></embed></object> </html>

Add comments below

Microtubules polymerized in H2O

<html><object width="480" height="390"><param name="movie" value="http://www.youtube.com/v/uEc3HmGloNo?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/uEc3HmGloNo?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="390"></embed></object> </html>

Add comments below

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