DNA Extraction

Don Lee, Presenter Author

10/10/2018 Added

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DNA extraction is an important first step in many molecular techniques. This video breaks the process down into its basic steps

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[00:00:08.030]DNA extraction
[00:00:10.110]is a regular, every day method
[00:00:14.440]that plant breeders use,
[00:00:17.430]and genetic engineers use.
[00:00:19.580]So we wanna take advantage of some learning here
[00:00:22.320]that's provided by an animation
[00:00:24.660]developed by a team called the Wheat CAP,
[00:00:27.280]under support by the USDA.
[00:00:30.340]And so let's take advantage of this animation
[00:00:33.630]and get a better understanding
[00:00:34.900]of how the DNA extraction process works with plants.
[00:00:39.310]So it's fairly easy
[00:00:41.590]to collect a sample of tissue from plants.
[00:00:46.760]And if we're interested in extracting the DNA
[00:00:50.740]from the cells within that plant tissue,
[00:00:53.800]we're gonna capitalize on both physical processes
[00:00:59.040]that help us get at that DNA
[00:01:01.370]and then chemistry that concentrates on
[00:01:05.200]some of the chemical differences
[00:01:06.940]between the DNA and the other biomolecule components
[00:01:11.490]in the plant tissue sample.
[00:01:14.600]Okay, so a lot of times the plant tissue is frozen
[00:01:20.180]or freeze-dried.
[00:01:22.520]This helps disrupt the cells,
[00:01:24.450]breaks open the cells
[00:01:26.950]and the DNA is of course inside the nucleus
[00:01:29.550]as a part of the chromosomes inside those cells.
[00:01:33.673]The more readily those cells are broke up, the better.
[00:01:36.890]So there's a number of methods that you can use
[00:01:39.580]to break up those cells,
[00:01:41.590]get 'em into small homogenized tissues.
[00:01:45.180]And then if you add an extraction buffer
[00:01:47.480]that's mostly water,
[00:01:49.250]that starts the extraction process.
[00:01:52.350]DNA is highly soluble in water
[00:01:56.370]and so by adding this extraction buffer,
[00:01:59.020]the DNA will start to go into solution.
[00:02:02.030]If we put it in a warm water bath,
[00:02:04.350]60 degree Celsius is pretty warm,
[00:02:07.530]the DNA will move into the solution at a higher rate.
[00:02:13.570]So after a short period of time,
[00:02:16.590]now we can start to capitalize on the difference
[00:02:20.320]in the chemistry between the biomolecules
[00:02:23.630]that are in the leaf that aren't DNA,
[00:02:26.690]like pigment molecules, and proteins,
[00:02:29.033]and the lipids that make up the membranes,
[00:02:31.920]and the nucleic acid, or the DNA itself.
[00:02:34.870]So we can add a chemical called chloroform
[00:02:37.990]and we can wash away those unwanted cellular components.
[00:02:41.330]We're adding the chloroform here,
[00:02:43.610]we are mix up the sample.
[00:02:45.870]Turns out the water and the chloroform
[00:02:48.630]have very different polarities and densities.
[00:02:51.120]So they don't mix very well.
[00:02:53.850]And so,
[00:02:55.840]if we do a little centrifugation,
[00:02:57.870]we can easily separate the water,
[00:03:00.370]the liquid that will have the DNA in solution
[00:03:02.940]with the chloroform,
[00:03:04.570]which will have a lot of other biomolecules in it
[00:03:08.580]like the green chlorophyll pigment, okay.
[00:03:12.030]So, now we've got both physical and chemical separation.
[00:03:16.910]So we can now concentrate on that top aqueous,
[00:03:20.710]or water layer,
[00:03:21.580]pull that off the tube.
[00:03:24.810]Add it to a fresh tube.
[00:03:27.030]Now what we want to have
[00:03:28.349]is the DNA to come out of solution, okay.
[00:03:32.290]If we add an alcohol like ethanol,
[00:03:35.300]especially if it's a little on the colder side,
[00:03:38.360]the alcohol and the water will mix
[00:03:40.400]but the DNA does not like to be in contact
[00:03:43.130]with the alcohol.
[00:03:45.100]And so the DNA molecules,
[00:03:47.120]when they're forced to encounter the ethanol,
[00:03:49.680]they'll come out of solution.
[00:03:51.050]We actually see a solid material precipitate
[00:03:54.410]inside the test tube.
[00:03:56.590]That's our DNA.
[00:03:58.070]Then there's a couple of different ways
[00:03:59.489]we could remove that.
[00:04:01.370]One would be to take a glass hook,
[00:04:03.470]if there was enough of the DNA,
[00:04:05.030]and just hook it out of there.
[00:04:06.810]Or you could,
[00:04:08.480]since you're working with a centrifuge tube,
[00:04:10.210]spin it again.
[00:04:11.970]And when you spin it,
[00:04:13.610]the solid DNA will form a pellet on the side
[00:04:19.110]or the bottom of the tube.
[00:04:20.950]So then we can just remove the liquid
[00:04:23.770]that doesn't have the DNA anymore.
[00:04:26.030]And then we can resuspend the DNA
[00:04:30.410]in a storage buffer,
[00:04:31.920]and we've got a sample of the DNA
[00:04:34.470]from that particular plant that we were testing.
[00:04:39.130]And now the DNA will be pure enough, clean enough,
[00:04:42.020]so that we can do molecular analysis techniques,
[00:04:44.590]such as PCR and electrophoresis.
[00:04:49.220]Okay, so this was Don Lee.
[00:04:50.774]I enjoy giving you this short presentation.

DNA Extraction

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DNA Extraction

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