User:Brian P. Josey/Notebook/2010/06/22

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Yesterday and Today

Today is going to be a busy day, I am fitting in what I was going to do yesterday with what I was planning on doing today. To that effect, I am running a PCR for Ant, creating a new emulsion, and filming and running a couple of magnetic experiments.

Emulsion and Magnetic Experiment

I needed to create a new emulsion because the other one would not go into the emulsion state like I wanted. I am following an old procedure that I stumbled upon a couple of days ago. To a small micro-centrifuge tube I added in order:

  • 1 μL Ferritin stock solution
  • 0.1 μL Tween 20
  • 98.9 μL mineral oil

I then sonicated the mixture until it became milky, and flowed in about 10 μL of the emulsion into a flow cell I created earlier. I noticed that today, it created an emulsion much more quickly than my attempt yesterday, taking only about 30 sec to reach the point I wanted. It appears that over time the emulsion mixture might break down and I'll have to replace it every week or so.

I put the flow cell on to the microscope. One thing that I noticed right away was that the droplets of ferritin were in a high concentration, and when I reach the point that I am measuring their velocity and plotting it as a function of their diameter and number of ferritin, I will have to have a lower concentration. This will prevent bumping, scrapping and any other interaction that would interfere with my data. For my experiment, I have the magnet held by a three pronged clamp so that it does not move and remains at a constant distance. I placed it so that the tip of the magnet was placed up against the glass as close as possible without interfering with the optics. This is what it looks like:

Magnetic Mount.JPG

I then ran two different experiments. One with the magnet being held on the upper right corner of the field of view, and then on the lower left corner. I ran each of these for 15 min, and took a picture of the screen every second. Here is the video for the magnet at the upper right hand corner:

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And the lower left hand corner:

<html> <object width="480" height="385"><param name="movie" value="http://www.youtube.com/v/Qs7koS-Dmc8&hl=en_US&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Qs7koS-Dmc8&hl=en_US&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385"></embed></object> </html>

In both cases, the frames are sped up so that while they were captured at 1 frame per second, they are presented as 10 frames per second. However, do to a precession issue in the software that I don't know how to resolve yet, it comes out to two minutes and not 90 seconds. This is the software that I use to assemble the video and not the capturing software. Note that these are not of the same field of view, but of two very closely spaced field of views. I had wanted to film the same spot, but the stage was bumped while I was reorienting the magnet, and I had to use two different field of views.

PCR

The same time that I was setting up and running the ferritin experiment, I was also setting up and running a PCR for Ant. The one that he asked for was similar to one he ran in November. He wanted 5 tubes containing the pRL574-F853-dig 10 μM dNTP, and five more tubes containing the pRL574-F834-dig 10 μM dNTP. All ten tubes contained the pRL574-R5263-bio 10 μM dNTP. Because we have new stock solutions of the chemicals, I had to recalculate the volumes added to achieve the desired concentrations. The final mixture of reagents came out to:

{{#widget:Google Spreadsheet

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Taking this, I then ran it on the Thermal Cycler for:

Stage Temp (C) Time (mm:ss) Reps
1 94 4:00 1
2 94 1:00
60 1:00
72 5:00 35
3 72 9:00 1