How to Precipitate DNA

MilliporeSigma and Seeding Labs Present: DNA Precipitation Hi, I’m Megan Kelly and today I’m going to show you how to precipitate DNA. This procedure is used to purify or concentrate nucleic acids from an aqueous solution. For this procedure, there are three main components that I’m going to tell you about. The first is salt. Today I’ll be using a 3 millimolar sodium acetate solution at a pH of 5.2. The reason why we use salt is because it neutralizes the
charge of the nucleic acid backbone, which causes DNA to fall out of an
aqueous solution. The next component is alcohol. The most widely used alcohol in this procedure is ethanol. It’s very important that you use at least a 95% solution of ethanol, and today I’ll be using 100%. Ethanol is used because it has a much lower dielectric constant as compared to water, which causes DNA to precipitate out of the aqueous solution. And the last component is temperature. Lower temperatures, such as 4 degrees, help promote precipitation of DNA out of the aqueous solution, and then this can be pelleted and recovered through centrifugation. Ok, let’s get started. Today I’ll be using salmon DNA at a concentration of 10 mg/mL. I’m going to take 100 microliters of the DNA and move it into an Eppendorf tube. Next, you want to add one-tenth the volume of your salt, so I’m going to add 10 microliters of 3 molar sodium acetate at pH 5.2. When you add the salt, there’s no need to pipette up and down. You can just very slowly add it to the DNA. And then if you want, you can invert the tube a couple times just to very quickly mix it. The next thing that we’re going to add is the 100% ethanol. So for here you’re going to add 3 times the volume that you have in your tube. So since we have about 100 microliters, we’ll add 300 microliters of 100% ethanol. Again, there’s no need to pipette up and
down. You just very slowly add it to the side of the tube and then you can invert it a few times. Now if you’re starting with a very high concentration of DNA, or you have large fragments, you should start to see your DNA precipitate out of solution. However, if you have low concentration or smaller DNA fragments, it’s unlikely that you’ll see anything here. Now we’re going to put the solution on ice for about 15 minutes, because as I said before, lower temperatures help promote precipitation of the DNA. So we’ll leave that in ice for about 15 minutes. Now after the 15 minutes has passed, you’re going to take your solution and you’re going to centrifuge it. Now if you have a centrifuge in the cold
room, or one that allows for temperature adjustments to 4 degrees, I would suggest using one of these. However, if you don’t have one accessible, then I would just use the one at room temperature. So you’re going to centrifuge for about 15 to 30 minutes at 15,000 x g. Once the centrifugation step is complete, you remove your tube, and as I said before, if you have a high concentration of DNA, or if you have larger fragments, you should see a pellet. However, if you start with a very low concentration of DNA, it’s unlikely that you’ll see anything here. Now it’s really important that when you remove the supernatant, that you’re careful not to disturb the pellet. So for this, I like to just very carefully dump the supernatant onto a paper towel and lightly dab. This way the pellet isn’t in any way disturbed. Next, we’re going to wash the DNA, and this step is just to remove any residual salt that may be remaining. So for this you can use a 70% ethanol, and I would add 1 mL to your tube. Again, you don’t want to pipette up and down. You just want to very carefully add it to the side of the tube because you don’t want to disturb the pellet. Then if you have access to a centrifuge at 4 degrees, I would use that. Otherwise, you can spin at room temperature at 14,000 x g for about 5 to 15 minutes. Once the centrifugation is complete, again you want to remove the supernatant, and again you want to be very careful. So I would just very slowly and carefully dump out the supernatant. Now it’s critical here that you let the pellet dry completely. You don’t want any residual DNA remaining. I typically place my tube in a biological safety cabinet and let it air dry. Once you’re certain that it’s completely dry, you can add your appropriate buffer and now you have purified DNA. Thank you very much for

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