User:Anthony Salvagno/Notebook/Research/2009/12/09/Practicing Unzipping
Things To Do
- Make new antidig
- Check components to make sure they are all new
- good sonication of beads
- Dilute DNA 1:10
- check - I accidentally used my digested pBR322 for this instead of the tetherable product, but then I fixed it.
- Reacquaint with tweezers
- Try to shittily tweeze.
Note from yesterday
I know it has been a problem in the past, but I don't think drift was a problem yesterday. It didn't look like anything had shifted between yesterday and the previous time of usage (which was a while ago).
I'm going to try Koch's humidity chamber method. This method is to follow the tethering procedure, but during the incubation times you put the sample chamber in a box filled with water. This way the humidity in the chamber will prevent evaporation of the sample. To make this, I am using an old pipette tip box. I poured a little bit of water in the box and will place my sample on the level of the tips (to provide separation between the water and the sample) as the water will sit at the bottom of the box. I am now leaving the box out for a little while to allow the water vapor pressure to build in my box. I'm fearful the box I'm using is inadequate for this job, but it is worth trying. At the very least it will be as bad as leaving it out in the room.
- I wonder if washing after the DNA step is important. My thinking is why not just try and let all the DNA remain in solution. This will give untethered molecules extra time to tether. Any attaching of free DNA to free beads will be wiped away in the washing phase after bead addition. I think I will try this today.
- Would it make sense to flow anti-dig more than once? What if an experimenter flowed a-dig once waited 5 min, and then flowed again and waited 5 more min.
- I am going to seal the sample and then go get lunch. Upon completion of this task, I will then bring my sample over to the tweezers and have some fun. Hopefully movie is on its way.
- Ramalldf 14:06, 14 December 2009 (EST):I think that it is all fair game as long as you can keep track of what you're laying on there (ie. the concentrations are what you expect them to be) so that it is easy to replicate in the future. The only problem having too much anti-dig on there that I could think of is that the you could possibly have more than one DNA stuck to a bead and not be sure that you're looking at an event due to only one DNA molecule. Would this hypothesis be applicable to your experiments? That's usually the response I get from people when I ask if I can ramp up the concentration of tethered protein to attach to more beads.
- Anthony Salvagno 17:46, 16 December 2009 (EST):That makes a lot of sense and I agree now that I think about what you said. Thanks for helping me see the light.
- Ramalldf 05:32, 17 December 2009 (EST): I think your idea about saturating the cell is good for positive controls. When things aren't looking too promising (ie. not seeing anything stick) I use concentrations that are larger than what I would use for acquiring data just to doublecheck that some other component of the assay isn't responsible for the lack of tethered DNA or protein. If I see some things stuck then I gradually decrease the DNA/protein concentration to acceptable levels. If I don't then I know something else is wrong with my assay and I end up having to remake some components of my assay :P
- Steve Koch 11:05, 17 December 2009 (EST): Thanks, Diego! Double-tethers are indeed undesirable. The concentration of antidig is actually really high 20 ug/ml is something like 200 nM. Who knows how many of those are "good" (it's polyclonal) or how many remain active once attaching to the surface. My intuition is that the 20 pM DNA limits tether density. But it'd be worth checking, say with 10x antidig. (BTW: If you want higher surface concentration of antidig, I think using higher concentration is better than multiple flows...also just a guess.) Finally, there's something very convenient about DNA unzipping: the sequence-dependent unzipping force is very well predicted and repeatable. Thus, when you do get occassional double-tethers, it's completely obvious: the force is twice as high until one of the tethers breaks! So, it's not as risky as many other assays where you can't nearly as easily tell from the data whether it's one or two). I wonder if there's opportunities to improve some of those other assays by including a little bit of unzippable DNA in series to act as a double-tether sensor?
I saw a few tethers and I messed with the Tweezers but I didn't take any data because I don't really remember how to do that. I'll need more Koch guidance. I did take some movies of me fucking around though and I have one interesting one about the laser power fluctuations that I'll need to edit down (tomorrow when I have more time). I think I will also try tethering with undiluted DNA to see if I get better tethers.