User:Anthony Salvagno/Notebook/Research/2010/02/10/Tethering, Now with more cleaning!

So I will try to tether once again. This time I will just do .51um beads sample and a control sample. I think my control last time was no DNA, but I think I should do no anti-dig. Today I will also clean my slide prior to use.

Slide Prep

I filled the sonicator halfway between min and max with water. I then poured a little bit of alconox into the water and stirred it up. I then put in one glass slide and one cover slip. I set the sonicator to 480 seconds. I rinsed with purified water 4 times and then blow dried. I then added tape to make the chamber and stuck it together.

Bead Prep

This time I had successful splashing. I rinsed and cleaned the sonicator after the above cleaning of the slides (to remove surfactant). I then filled the sonicator up to just above the min line with water. I did a 1:25 dilution of beads to BGB (in Pop 1x) and put it in one of the small tubes (the 0.2ul ones I think). I set the sonicator to 180 seconds (default) and I immediately got splashes. I submerged the tube more than halfway or at least until my fingers just barely didn't touch the water. Oh yea and I held the tube by hand. I had to spin it down afterward, which I hope isn't counterproductive.

Tethering

{{#widget:Google Spreadsheet |key=tDh4kF8jsFVb9T7nmjuWEaw |width=500 |height=300 }} For the control I am deciding to do no DNA instead of no anti-dig. Although I think tomorrow I will do a two controls side-by-side to see if there are any differences. I am using Brian's 1.1kb dsDNA tether. It is diluted 1:100.

Results

At first I was worried, because I had forgotten which sample had the DNA and which did not. When I looked through it was obvious. Can you pick it out:
Sample 1

• stuck beads - about 1 or less per FOV
• I saw 2 jigglers the entire time. One was stuck to another bead and the other I couldn't tell. There were definitely other jigglers but I discounted those because I was getting near the nail polish. I saw the banding effect I mentioned yesterday and so I limited my scope to the middle of the sample away from both ends.

Sample 2

• There were about 10 stuck beads per FOV.
• Some FOV's had as many as 20 stuck beads, but that only happened 2 or 3 times.
• There was at least 1 jiggler in every FOV, and the ones that had upwards of 10 beads had at least 3 jigglers
• Stuck:Jiggler Ratio was about 4:1
• I saw no floppy jiggles either. All were in the range of about .5um motion diameter.

I don't know what the difference between today and yesterday is, but it seems like I should clean the glass prior and not way prior.

UPDATE: I just found these two petri dishes that were labeled precleaned glass and precleaned cover glass (respectively). This means that I did not make sample chambers with the cleaned glass. So last time's was just dirty ole glass. Tomorrow I will try to tether with my precleaned glass making new sample chambers from one set. This will let me know if I can clean a bunch of glass and use it later.

Other

HAHAhAHA! I can edit this from within an iframe! Woo-hoo. I've been wanting something like this.

Steve Koch 21:57, 10 February 2010 (EST): Looks like nice result! A lot stuck, but could be because of too much DNA? Can you estimate how much DNA? It's not clear from above what concentration of DNA was. Also, I cannot see the iframe thing? Maybe you mean that you iframed this page on some other page?

Anthony Salvagno 11:03, 11 February 2010 (EST):I framed this page on another page (my sample notebook).

Steve Koch 09:27, 11 February 2010 (EST): Also, seems like you're getting good at tethering, so doing many more today would be good. Perhaps also in the context of showing Pranav how to do various things. In addition to the clean glass, another study to do today would be DNA dilutions. You can make a plot of # stuck beads and # jiggling beads as a function of DNA concentration.