In this video, we're going to talk about the southern blotting technique. And so southern blotting, again, is a specific technique that's going to be used to rapidly detect a specific DNA sequence by using DNA probes. Now there are other types of blotting that exist. For example, northern blotting, which is going to be used to detect RNA sequences, and Western blotting, which is going to be used to detect specific proteins. But again, in this video, we're going to be mainly focusing on southern blotting, which is used to detect DNA sequences. And so down below, you can see that we're showing you the 5 different steps of southern blotting that are numbered 1 through 5. And of course, these numbers that you see here in the text, 1 through 5, correspond with the numbers that you see down below in the image 1 through 5. And so here in the first step of Southern blotting, we're going to take some unknown DNA samples and fragment them, and then separate those DNA fragments within the unknown DNA sample by size by using gel electrophoresis, which we covered in previous lesson videos. And so if you take a look at our image down below at step number 1, what you can see here is that gel electrophoresis is going to be used to separate fragments of unknown DNA samples. And so you can see our gel electrophoresis down below here. And so in step number 2, what we're going to do is take the DNA on the gel and denature the DNA on the gel so that the DNA on the gel becomes single-stranded DNA or ssDNA, for short. And the way that the DNA is going to be denatured to single-stranded DNA is by incubating the gel with a denaturing buffer. And so if we take a look at our image down below, we can get a better understanding of this. And so what happens is this gel from step number 1 is going to be placed into a container that has multiple different pieces in it. First, this container is going to have the denaturing buffer that's going to help denature the DNA and create single-stranded DNA. But there's also a sponge that's going to be on here. The gel is going to be placed on top of the sponge. Then you'll see a nitrocellulose filter paper which is here in a greenish color, is going to be placed on top of the gel. And then on top of the nitrocellulose filter paper, we have the paper towels that are going to be placed on the very top. And so basically what happens is, the denaturing buffer is going to be absorbed upwards, through all of these different components. And so the denaturing buffer is a liquid that's going to be absorbed into the sponge, which is going to migrate and be absorbed into the gel, which will migrate and be absorbed into the nitrocellulose filter paper, which will then continue to migrate into the paper towel. So basically, the buffer, the denaturing buffer is going to be migrating upwards as you see here in this image. Now the denaturing buffer, the way that it denatures the DNA and creates single-stranded DNA is because the pH of the buffer is actually going to increase, the pH. So the pH is going to be increased, and that causes the DNA to be denatured to single-stranded DNA. Now the filter paper is going to be used to blot the gel, and the blotting here is really just referring to transferring the DNA from the gel to the filter paper. And so it says here that the filter paper will be used to blot the gel and absorb the denaturing buffer. And the denaturing buffer is going to migrate all the way through all of those substances to get to the paper towel stack at the very top. Now, again, what this is going to do is transfer the DNA from the gel to the nitrocellulose filter paper. And the DNA again is going to be denatured as single-stranded DNA. So if we take a look at step number 3, you can see that the single-stranded DNA is transferred over to the nitrocellulose filter paper as the denaturing buffer is being absorbed and migrating through to the paper towels. And so, ultimately what happens is, you can see that the single-stranded DNA fragments are now found on this nitrocellulose filter paper, which we can grab the nitrocellulose filter paper and transfer it to another container here. And the gel, notice, no longer has any more DNA in it because all of the DNA has been denatured to single-stranded DNA and transferred over to the filter paper. Then in step number 4, the filter paper is going to be removed and incubated with radioactive probes that are complementary to the specific sequence of interest that we want to try to detect. And so if we take a look at step number 4, what you can see is we take the nitrocellulose filter paper, and we can put it into a container. Here, we're just showing you a little ziplock baggy, and expose the nitrocellulose filter paper to these DNA probes. And so the nitrocellulose filter paper is going to be incubated with these DNA probes. And recall that the DNA probes are going to be radioactive and they're going to be complementary to the sequence of interest. And so they will bind only to specific fragments of DNA that, it is complementary to. And so in step number 5, the final step here, the radioactive filter paper can be analyzed and visible bands on that filter paper are going to be the ones that bind to the DNA probe in step number 4. And so if you take a look at step number 5 down below, you can see that the DNA is going to be complementary to is the DNA complementary to the probe is visualized. And so what you'll notice is that here we only have a specific set of bands, right here and right here. These bands are the ones that are complementary to the probe. So notice over here on the left-hand side, there are a lot more DNA bands in the filter paper. But the only ones that will actually be visualized are going to be the ones that are complementary to the probe. And so these DNA bands here that are in yellow, these are the only DNA bands of all of the DNA bands that were over here that are actually complementary to the probe. And so these are the bands that are going to have the sequence of interest. And so we can say that lanes number if we were to number these lanes, 1, 2, 3, and 4, lanes 2 and 4, right here and right here are going to be samples that actually contain the sequence of interest that the scientist is interested in. And so this is basically how southern blotting works. It's used to detect specific DNA sequences that are complementary to DNA probes. And so this here concludes our brief introduction to southern blotting, and we'll be able to get some practice applying these concepts as we move forward in our course. So I'll see you all in our next video.
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Southern Blotting
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Southern Blotting practice set
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