User:Brian P. Josey/Notebook/2009/12/08

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DNA Tethering

Originally, I intended to create a page on my notes on DNA tethering, however we needed to have some tethers today. I am making them, and as I will go through the process, I will include my notes on the procedure, and the procedure that I am doing as I go through it. For today, I am using sample 3B from Thursday as my DNA. The concentration that I made on Thursday is too high, so for today I diluted it down to 1:100 of the original concentration. To do this, I diluted 1 μL of the original sample with 99 μL of DI water. The process is:

  1. Create a Flow Cell You need a flow cell that has a flow chamber volume of about 10 μL.
  2. Anchoring Flow in 10 μL of anti-dig and then wait 3 minutes.
  3. Surface Blocking Flow in 50 μL of BGB/Pop solution and then wait 3 minutes.
  4. Tethers Flow in 10 μL of the tethers and then wait 7 minutes.
  5. Wash Flow in 50 μL of BGB/Pop without a wait time.
  6. Beads Flow in 10 μL of the beads and wait 10 minutes.
  7. Wash Flow in 50 μL of BGB/pop without a wait time.
  8. Seal Flow Cell Use some nail polish to seal the flow cell as soon as you are done with the final washing.

For the above procedure, a volume of chemical and a wait time are both given. When you are flowing in the new chemicals you can't flow the entire volume in all at once. So what you want to do is place a small part of the chemical on one side and use a wick to get it to flow across the cell. When you do this you want to be careful to not let air bubbles form in the flow chamber. To do this, you want to leave a small amount of liquid on both sides of the flow cell, which you want to leave out when you are letting it settle. This part of the process is hard to explain, and is something that you really have to be shown to know how to do well.

DNA The DNA here will be connected to the glass by the anti-dig, and will be attached to the beads. For this particular tethering protocol, I used the 3B sample from Thursday because it seamed the most promising. It has the combination of the pRL574-F853-dig and pRL574-R2008-bio primers. The concentration of the DNA can't be too high, and the concentrations that come from PCR are way to high, so you want to dilute it. For this one I diluted it down to 1/100th of the original concentration by mixing 1 μL DNA in 99 μL of DI water.

Anti-dig Anti-dig is an antibody that will bind to the dig and attach to the glass slide, cover slip, and probably the tape. You flow it in first so that it can attach to the glass at random, giving many good spots that tethering can be done at.

BGB/Popping Buffer This solution has three purposes. The first is to wash away any excess DNA, beads or whatever that isn't needed, as in steps 5 and 7. The second is that the BGB will, ideally, settle on the glass wherever there isn't any anti-dig preventing any exposed glass in the flow cell. Finally, the popping part of the buffer will help remove any proteins from the strand of DNA when tweezing.

Beads The beads are used during the tweezing to hold the DNA in place as it is pulled apart.

This table was made by Anthony and reiterates the tethering process:

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