Jason Williams, DNA Learning Center, shows how to precipitate DNA.
At this point your samples have been incubating for about 30 minutes: 15 minutes at 65 degrees and another 15 minutes at 37 with the RNase solution. When you remove the samples from the heat block or the water bath, you may notice some color change from the oxidation, and of course at this point the nuclei, the chloroplasts, the mitochondria, they’ve been lysed by the solution, and we’re going to add protein precipitation solution.
This protein precipitation solution will take the proteins that are soluble, that are in the liquid right now, and force them to come out so that we can get rid of them when we actually want to extract the DNA. Because the protein precipitation solution can be potentially a little bit of an irritant, we can wear goggles at this point. Then you’re going to add 200 μL of the protein precipitation solution to each one of the tubes. When you add the solution you may notice a little bit of a color change; the solution originally is clear, but as you leave it and put it on the ice, it may become milky, so just pay attention to that color change when you see it. Once you’ve added the 200 μL, put each tube on ice and leave it there for about 4 minutes.
Now what you want to do is take the samples and put them into your centrifuge; and when you put them in, try to make sure that the hinges are always facing outwards, that way the pellet that forms will be on the same side as the hinge and it’ll be easy to see. Put all your tubes in in a balanced configuration; you can spin this down for 4 minutes.
Once your samples have finished spinning, remove them from the centrifuge, and you’ll notice that all of the cellular detritus has lodged itself on the side of the wall. So what we’re going to do now is remove the liquid supernatant and leave the rest of the material behind. You need to have labeled clean tubes, and so we’re going to transfer 600 μL of that supernatant from this tube into our freshly labeled tube. When you go to take the supernatant out of this tube, you want to be careful to avoid sucking up as much of that debris as possible. So take your pipette, slowly go in, and remove that liquid supernatant, leaving most of that material in the bottom there. Take that supernatant, transfer it to the new tube, and then you’re ready for the next step.
The next thing we’re going to add is the alcohol; in this case we’re using isopropanol. You’re going to add 600 μL of isopropanol to each one of your tubes. Take up the 600 [μL], and then if you add it slowly to the tube, letting it drip down the side, you’ll get a little bit of a layer because the isopropanol and the other liquid in the supernatant don’t mix right away. Sometimes at the interface of the isopropanol and the layer that’s there, you can see a little bit of the DNA precipitating out of solution. The next thing that you want to do is actually take this and invert it several times, just up and down, and after you do this for a few minutes you can actually see the DNA precipitating out. Once you’ve added the isopropanol to all of your tubes, take them and spin them down for 1 minute at maximum speed.
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