User:Anthony Salvagno/Notebook/Research/2010/02/12/DNA Concentration affects during Tethering

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Background

This is going to be a To be continued... kind of post because I can see doing this many times for some decent data. Anyways I think it is a great idea to see how the [DNA] affects tethering, bead sticking, etc. Yesterday I purified some PCR DNA from a reaction Brian did in December:

This will be my starting concentration.

Setup

I will dilute the DNA several times in this fashion:

  • Starting amount
  • 1:5 dilution - 2ul start in 8ul water
  • 1:10 - 1ul start in 9ul water
  • 1:20 - 0.5ul start in 9.5ul water
  • 1:50 - 1ul 1:5 in 9ul water
  • 1:100 - 1ul 1:10 in 9ul water
  • 1:500 - 1ul 1:50 in 9ul water
  • 1:1000 - 1ul 1:100 in 9ul water

I don't think I will get to try all these dilutions today. But I will tethering with the most concentrated amounts and work my way down and continue next week.

Tethering

View/Edit Spreadsheet

I sonicated the beads this time for 15 seconds. The splashing inside the tube was immediate as in as soon as the tube hit the liquid, there was splashing. After about 5 seconds there was significant splashing and I gave it an extra 10 seconds for good measure. I am not spinning it down this time in case the spinning allowed for clumps. I am also going to try my precleaned glass.

Normally I wash with BGB after each step and I use 50ul (5 sample volumes) to perform the wash. Here I used 10ul for one wash because I was running low. I am doing it after the DNA stage because I think that is where it is the least crucial to wash out of the 3 wash points (not that they aren't all important). I am doing 10ul instead of nothing because I think it is important to get some untethered DNA out especially in the high concentration sample.

Results

I'll be looking for number of stuck beads per field of view (FOV) and number of jigglers compared to number of stuck beads. I will also look at the jiggling range (more DNA means more tethers can attach to a single bead so beads could jiggle less at higher concentrations). I'll try and post images of each sample here.


Steve Koch 22:25, 12 February 2010 (EST): Very nice notebook page and good experiments. Do you think the "floppy" tethers are the good ones? It does seem, based on your evidence, that the undiluted samples are too concentrated. Looks like 1:5 is a good bet.
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