User:Anthony Salvagno/Notebook/Research/2010/02/08/Planning for Tethering Practice

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I had been having tethering problems for a while and then I looked over some old notes of Koch's and corrected many of the problems I had been having and it worked. Moral of the story: don't make shit up when you are doing an experiments. Apparently I had been playing telephone with myself and somehow I had changed the protocol to be the conglomeration of mistakes I had been making. Now that hopefully all that has been corrected it is time to experiment in preparation for the rush of getting shit done by BPS. I will be referring to this page for plan accurateness.

Contents

Supplies

  • Anti-dig
  • BGB
  • DNA - I will practice with my TpBR and when it is time for unzipping, I will use my T14 clones.
  • microspheres - I want to try and achieve tethering with both our .51um spheres and our .97um spheres
  • glass slides and coverglass

Protocol

Cleaning Slides

Last time I cleaned a bunch of slides and coverglass. I sonicated all the glass in alconox and water (The alconox was 1% starting and diluted further in the bath). I sonicated for 4 min. I also had prepared a bunch of slides and coverglass at one time. I can't quite remember (and I didn't write it down), but I think I used the remaining glass (not used that day) to make microchambers for future use. I do have a box with a bunch of premade stuff so I will use some of that and some freshly cleaned glass for my experiments. Sonication in alconox solution seems like a good plan. Here is what I will do:

  • Clean glass at least 5 min prior to use, bu not more than 30 min prior to use.
  • Use soluion of alconox and water in sonicator.
  • Sonicate for 4 min
  • Rinse thoroughly (4 times or more)
  • Blow dry

Make BGB

I need 5ug/ml BGB in popping buffer. Usually measure out the required weight for 3ml of pop, but already have some BGB made. I will use what I have left and then make a larger batch if needed.

Prepare Anti-dig

Dilute 1:10 in PBS.

Prepare Microspheres

Dilute about 1:25 in BGB and make samples prior to tethering. Previously I had been making about 100ul aliquots and the beads would settle after a day or so and clump. To prevent clumping, I will just make aliquots for immediate usage. Also I will give the beads a good sonication as well since they have a tendency to clump after addition of BGB (they don't clump terribly though). Since I will be doing a few experiments 25ul aliquots of both types of beads should be ok to start and I will make more as I go.

Tethering Procedure

Since tomorrow is a short day for me (because of classes) I will use mostly already prepared stuff. I should have:

  • BGB
  • anti-dig
  • DNA
  • glass

So I will just need to prepare my beads.

Typically my procedure is:

View/Edit Spreadsheet

I will do two experiments at first. Each will be a dual sample slide meaning I will have two channels on each slide and I will use 2 slides (one slide per experiment). One channel will have DNA and the other channel will contain no DNA acting as a control. Some of the things I look for in each sample:

  • tethered particle motion and amount of motion - truly tethered beads will have a range of motion of about half a micron
  • stuck bead amount - lots of stuck beads indicate a problem
  • ratio of stuck beads : moving beads - lots of stuck beads and low tethered beads means poor tethering efficiency

I basically want to look at as many field of views as possible to gauge success of experiments.

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