User:Anthony Salvagno/Notebook/Research/2010/01/20/Tethering Practicing

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I need to get tethering working well and I need to get it soon. There is a lot of variables and I need to break it all down. I will do Koch's protocol exactly and if I can get that working, then I will alter it to make it easier. First things first.

Contents

Koch's Tethering Notes

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Cleaning Slides

Since I don't have windex I will sonicate my slides for a few minutes. This should work.

  • Andy Maloney 00:35, 21 January 2010 (EST): We really need to find some way of cleaning our glass slides such that we know it's clean and it's reproducible. Maybe we can ask Brian to research how people clean their glass.

Anti-dig

I have learned that PBS is what I want to use to dilute my anti-dig. I think I've been using Popping buffer. Maybe this has been a problem.

Beads

Also of note, Koch mostly made a dilution for immediate use in BGB. I will do this and then sonicate for a couple of minutes.

My Exact Tethering Protocol Try 1

Preprep

  • Ok I am cleaning the slides in water with 1% alconox. I filled the water about halfway between min and max lines and then added some alconox to it (maybe making it up about 3/4 of the way). I left the sonicator on for 380 seconds. The coverslides float so I hope this isn't a problem. After sonication I rinsed a bunch of times in the little basket with our pure water. I rinsed 4 times and blow dried.
  • I did a 1:20 dilution of the beads in BGB. I will sonicate for 180 seconds or until I see splashes.
  • I made a 1:10 dilution of anti-dig in PBS.

Fluidics

View/Edit Spreadsheet

Results

This worked out swimmingly. I think the PBS in the anit-dig might have been the problem. The sonication definitely worked (for the beads) and having clean glass was nice. In both samples I saw very few stuck beads. In the no DNA sample there were significantly less stuck beads. I think I counted about 4 stuck beads per FOV, maybe less. In all the FOV's I saw, there was only a total of 3 jigglers and I must have looked at 20 FOV's. In the DNA sample the first FOV I looked at there were 5 stuck beads and 9 jigglers. The rest were on the order of about 4:3 (stuck:jiggler) and not many of them were loose. In that first FOV, I think of the 9 jigglers only 1 was floppy as in more motion then a tether could have. I also didn't see any tight motion indicating multiple tethers. I will try another one after the gym.

Try 2

I ran out of time today because of preplanning for project Jasper. I will do more on Friday at the latest (classes and project Jasper work tomorrow).

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