DNA Barcoding Protocol: Isolating DNA

Jason Williams, DNA Learning Center, goes through the steps involved in isolating DNA from an animal or plant sample.

Equipment, Reagents & Safety We’re going to take the samples that we prepared earlier and extract the DNA. In order to do this, you’ll need the following pieces of equipment. First, you’ll need pestles, which we’ll grind the samples with; scissors [or] forceps – you can use those in order to actually get a smaller piece of that sample. We’ll need micropipettes, and here I have the three sizes: the P1000, which holds from 100 to 1000 μL; the P100, which holds from 10 to 100 μL; and the smallest one, our grey pipette, the P10, which holds from 0.5 to 10 μL . And of course I have the appropriate tips for each size. For the pipettes of course you’ll need a waste beaker for the excess pipettes, and it’s helpful to have some napkins lying around for our DNA isolation step. You’re going to need a tube rack along with some 1.5mL microfuge tubes; you need 2 tubes for each sample that you’re going to be preparing. For the larger equipment in this step you’ll need a heat block or a water bath. You need to have a heat block set at 65 degrees and one at 37 degrees. If you only have one heat block just allow yourself enough time for it to cool in between steps; if you have a water bath it’s easy to change the temperature. We’re going to start at 65 degrees, so please set your heat block at this time. You’ll also need a microcentrifuge in order to spin your tubes down. One of the most important things you’ll need is a notebook. You want to note all of your samples, give them a number and ID – I’m just calling mine sample 1, 2 and 3 – note who collected them, note where they were collected, as well as any other information that you require. It would also be helpful to have a copy of the protocol to follow along. As far as chemical reagents you’re going to need the nuclei lysis solution. This you can already have on ice, and I’m going to put it on this beaker of crushed ice that I have here. You’ll also need the protein precipitation solution, the RNase solution, and at the last step you’ll need the DNA rehydration solution. When we actually precipitate the DNA we’re going to be using isopropanol, so you’ll need a tube of that, as well as 70% ethanol. Now, you may already have your chemical reagents already aliquoted into smaller tubes; I have the full sized stock versions. Whatever you have is fine, just be sure to follow the protocol so that you use the proper amount for each step. All of the chemical reagents that we’ll be using in this protocol are extremely safe, non-toxic [and] non-hazardous. However, it’s always good scientific practice to use personal protective equipment. So, if you choose to, you can wear a pair of latex gloves, just be sure to change them frequently in order to avoid cross-contamination of samples. For the protein precipitation step, since the chemical can be a little bit of an irritant you may choose to wear goggles, and that’s what’s recommended so I’ll be wearing that during that step. Step 1: Preparing the Sample To begin the protocol, first you need to get a sample of the individual things that you’ve collected. In this case, I have a broad leaf from a maple tree, I have some salmon that’s here, and I have some insects, which are probably crickets. So, what I’ll need is one tube that I’ve labeled for each one of my samples. When you label your samples, and I’m just going to use a Sharpie, give them the designation – I’m calling this one Sample 1 – I’m giving it a date that I’ve actually collected it on or processing it on, and I want to label it on the side as well as the top of the tube just so I can always be sure. Now when you collect your sample for DNA extraction or when you actually take a small piece of it, less is actually more. It’s actually very important to get a small amount of the sample. For a leaf what we can use is actually a blue pipette tip; this is a P1000 tip, and we can actually use it to make a cutout of the leaf, and really what you’re going for is a very, very small section, so I’m just going to take that. And when you use the tip you actually get a nice round cutout, and I’ll use the forceps to grab this out of the pipette tip. Now working with fish or other things, such as insects, you’re looking for about 10 to 20 mg, which is a very, very small amount of sample. To illustrate with this piece of salmon, I’m just going to take the scissors, take my forceps and actually cut off a very, very small amount. It’s a little bit less than the size, perhaps, of a pencil eraser, and it’s just that amount that you need in order to get quite a bit of DNA from. I’ll take this, put it into my tube, and of course I want to make sure that I label my tube so that I don’t forget what sample this is. In some cases, when doing DNA barcoding, you’re actually going to be the author of the barcode. What that means is that not only did you collect DNA and sequence it in order to obtain the DNA barcode sequence, but that you preserved the specimen so that other scientists could study it. What that means is that when you’re collecting a specimen like this cricket, you’re not going to damage the whole insect; you actually want to take just a small amount so that it’s actually identifiable by traditional taxonomy methods. So in this case, we’re just going to take a leg from this cricket, and the rest we can pin and label and store so that we can be the author of the barcode. Here I have a small amount, and that’s really just enough for us to do the DNA barcoding with. Step 2: Extracting the DNA Now that you have your samples in their individual and labeled tubes, you’re ready to begin with the extraction. What we’ll need to do is add 100 μL of the nuclei lysis solution, so I’m going to use my blue pipette set to 100 μL, and always I want to use a fresh tip to add the solution every time. My lysis solution is already on ice, so I’ll carefully get 100 μL, cap that back up, and add that 100 μL to my first sample. Once you’ve added the solution to the sample, you can take a clean pestle and start grinding the sample forcefully for about one minute. Once you’ve ground the sample thoroughly, and in some cases you may notice that the solution changes color – chlorophyll might be released with plants, with animals you may see some color change – just grind it until you hopefully see some of that color comes out; that means that you’ve done a good job. Then add the remaining 500 μL of the nuclei lysis solution. Make sure to mix after you’ve added that last 500 [μL]of the nuclei lysis solution, and add this to the heat block. Once your samples have incubated for 15 minutes at 65 degrees, you have to add the RNase solution. The RNase solution contains RNase enzyme, and that enzyme is going to actually break down a lot of the RNA that’s present in the solution, so that when we actually precipitate nucleic acids, the majority of what we’re going to get back is DNA. I’m taking my P10, and I’ve set it to 3 μL, and you want to take a fresh tip, take up 3 μL of the RNase solution, and add that to each one of the tubes. Once you’ve added it to the tube, you want to shake the tube and then place it at 37 degrees; so if you have another heat block place it in that one, if you need to let this heat block cool just cool it to 37 degrees and then place this to incubate for an additional 15 minutes. Step 3: Precipitating the 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. Step 4: Washing & Eluting the DNA Once your tubes have finished spinning, you can remove them from the centrifuge, and you should notice a pellet that’s on the same side as the hinge, and that pellet is actually the DNA that’s come out of solution. What you want to do now is carefully remove and pour off the isopropanol. So open the hinge, and over your waste beaker just carefully pour off that alcohol. If you have a drop or two remaining, you can use a napkin to just get that last drop, but it’s important that you don’t shake the tube so much. When you remove the ethanol or the isopropanol, when we’re doing these alcohol precipitation steps, that’s when the pellet is most visible, so if you don’t see it or it’s very clear previously, when you don’t have those alcohols it’s easy to see. At this point, go ahead and take your tube and add 600 μL of the ethanol; the ethanol just washes away any excess impurities. Once you’ve done this to all of your tubes you can shake them a bit and then spin them down for 1 minute at maximum speed. After you’ve added the ethanol to all of your tubes and you’ve spun them down, the next step is to take your samples, and again carefully pour off any excess ethanol, and again you can use a napkin to get rid of anything else that’s remaining, just gently tapping it. You want to make sure that the pellet that was there in the last step is still against the side of the tube. And let the tube sit open on a rack for about 10 to 20 minutes to let it air dry. If you have a hair dryer set on a low setting, and not blowing directly into the tube but on the side, you can accelerate the drying process. After you’ve air-dried your pellets and you don’t see or smell any remaining ethanol, you’re ready to add the DNA rehydration solution. You need to add 100 μL, so I’m going to use a fresh tip to add 100 μL of that solution to each one of my tubes. Cap the tube, and you can use your finger to vortex a little bit and shake it up, and then place that on a heat block or a water bath at 65 degrees for about 45 to 60 minutes. If you’re going to use the DNA immediately, you can keep it on ice until you go to the PCR step; if you’re not going to use the DNA immediately, then you can put it at –20 until you’re ready to use.

DNA, barcoding, lab, protocol, isolate, isolation, specimen, sample, pipette, extract, extraction, precipitate, precipitation, equipment, reagents, centrifuge, identification, DNALC, CSHL, DNA Learning Center, Cold Spring Harbor Laboratory, high school, middle school, experiment, eluting, Urban Barcode Project, COI, rbCl, gene, genetic

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