Steps of Southern Blotting

by Jason Amores Sumpter
134 views
1
1
Was this helpful ?
1
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 D N a sequence by using DNA pros. Now there are other types of blotting that exists, 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 five different steps of Southern blotting that are numbered one through five. And of course, these numbers that you see here in the text one through five correspond with the numbers that you see down below in the image one through five. 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 gel electrophoresis and previous lesson videos. And so if you take a look at our image down below at step number one. What you can see here is that gel electrophoresis is going to be used to separate fragments of an unknown DNA samples. And so you can see our gel electrophoresis, uh, down below here. And so in step number two, what we're gonna do is take the d n a on the gel and denature the d n a on the gel so that the DNA on the gel becomes single stranded DNA or S S d n A for short and the way that the D N A is going to be denatured to single strand of d N A is by incubating the jail with a denature ring 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 one is going to be placed into a container that has multiple different pieces in it. First, this container is going to have the DNA featuring buffer that's going to help denature the d. N A and create single strand of 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. Uh, then you'll see a nitrocellulose filter paper, which is, uh, here in 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 gonna be placed on the very top. And so basically what happens is, um, the Denature ring buffer is going to be absorbed upwards through all of these different components. And so the DNA featuring 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 be then continue to migrate into the paper towels. So, basically, the buffer the DNA featuring buffer is going to be migrating upwards. As you see here in this image, uh, now the DNA featuring buffer the way that it d Nature's the D. N A and creates single strand of D N A 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 D. N a, uh, to be denatured and and denatured to single stranded DNA. Now the filter paper is going to be used to block the gel, and the blotting here is really just referring to transferring the D N A. From the gel to the filter paper. And so it says here that the filter paper will be used to block the gel and absorb the denature ring buffer. And the DNA featuring 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 what's going to happen is, um, the D n A. From the gel is going to be transferred to the nitrocellulose filter paper, and the D N A again is going to be denatured a single stranded DNA. So if we take a look at step number three, you can see that the single stranded DNA is transferred over to um, the nitrocellulose filter paper, as the DNA featuring buffer is being absorbed and migrating through to the paper towels. And so ultimately, what happens is you can see that the single strand of 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, Uh, and the gel noticed. No longer has any more d n a 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 four, the filter paper is going to be removed and incubated with radioactive probes that are complementary two, the specific sequence of interest that we want to try to detect. And so, if we take a look at step number four, 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 baggie and expose the nitrocellulose filter paper to these DNA probes. And so the nitrocellulose filter paper is going to be incubated with these d n A 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 five, the final step here, uh, the radioactive filter paper can be analyzed and visible. Bands on that filter paper are going to be ones that bind, uh, to the DNA probe in step number four. And so, if you take a look at step number five down below, you can see that the d N A. Is going to be complementary to, uh is, uh, the D n a. Complementary to the probe is visualized. And so what you'll notice is that here we only have a specific set of bands. Uh, right here and right here, And 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 uh, 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 a number of these lanes 123 and four lanes two and four 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 year 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.