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Southern blot

Southern Blotting is a technique used to identify specific DNA sequences in a complex mixture. It involves cutting DNA into fragments, separating them by size through gel electrophoresis, transferring them onto a filter, and exposing them to a radio-labeled DNA probe. This process helps visualize the presence of a gene of interest.
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Created by Ronald Sahyouni.

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Video transcript

- So in this video, I'm gonna be talking about something known as a Southern Blot. So, a Southern Blot basically allows you to visualize a specific piece of DNA that you're interested in. So let's imagine that we have a cup and it's filled with DNA. So it's got just a whole bunch of DNA inside. And there's just lots and lots of those DNA and let's imagine that I'm specifically interested in one gene. So let's imagine that I'm interested in Gene A and I want to see if Gene A is inside of this cup. If it's inside of this long piece of DNA. Now, in order to figure out whether or not Gene A is inside this cup, basically we have to do this process known as a Southern Blot. And we'll break it up into a couple of different steps. So Step 1, what we're gonna do is we're gonna take this DNA and we're gonna cleave this. So, "take the DNA and cleave it." So, let me draw that out. So, we're gonna take this big old strand. We're gonna remove it outside of the cup over here. So, we got this big strand and we're gonna cut it up. We're gonna expose it to enzymes that will basically cleave the DNA in a whole bunch of different parts. And that will result in lots of these smaller pieces of DNA. So that's basically the first step. So we got a bunch of small little pieces of DNA. Now Step 2, what do we do? Well, what we're gonna do is we're gonna take all these tiny little DNA fragments and we're gonna run them on the gel. So, specifically we're gonna do a gel electrophoresis, "electrophoresis" on these DNA fragments. And I made a video on gel electrophoresis if you want to refresh, you can watch that video. But basically, the gel electrophoresis will help us separate these DNA fragments based on size and based on charge. So, let's just diagram that out. So, we're gonna take these DNA fragments and we're gonna run them on a gel. So, let's imagine that this is the gel and we add the DNA fragments to different wells. So the fragments are gonna move down the gel and they're gonna basically be separated based on size and based on charge. So, we're gonna have these fragments separated like so. So now, we've got this gel and we've got the DNA fragments separated by size on this gel. So the next step, step number three is basically we're gonna take this gel and we're gonna transfer it to a filter. So, transfer the gel onto a filter. And what the filter will basically allow us to do is it allow us to visualize 'cause this gel is very flimsy. So, we want to transfer it onto a filter. What we'll do is we'll take a filter that's basically the same size as the gel and we're gonna basically just put it right on top of the gel for a little bit and the fragments will basically transfer on to the filter. So now, we're gonna have a filter with these fragments and the filter is a lot sturdier than the gel. So this is the filter and I'll just write that down over here and this over here is the gel. Okay, so the next step, step number four that we're gonna take the filter and we're gonna expose it to a radio-labeled the piece of DNA. So, "expose to radio-labeled DNA." Now, this radio-labeled DNA is going to be the complement to our gene of interest. So, we're interested in finding out if Gene A is present in this mass of DNA over here. So what we do is we're gonna take the complementary sequence to Gene A and radio-label it and expose it to this filter. So, let's imagine that the radio-labeled piece of DNA is this pink piece of DNA. And let's imagine that we do have Gene A, so let's imagine that this piece of this DNA fragment was actually Gene A or our gene of interest. So what's gonna happen is when we expose the radio-labeled DNA to this filter paper, it's going to anneal to our gene of interest. So we're gonna have this radio-labeled piece of DNA stuffed to this DNA fragment which it's complement. So, in order to visualize it, in order to visualize this radio-labeled piece of DNA, we have to do the fifth and final step which is expose the filter to an x-ray film in order to visualize the radio-labeled probe. So, "expose to x-ray." And the x-ray basically it will shoot a bunch of x-rays and since this piece of DNA is radio-labeled, it will pop up on the x-ray film. So, we're gonna have a film and we'll draw that film over here so we'll have this film and basically the only thing that will pop up is this fragment over here and that fragment will have a control and we'll be able to say, "Okay. Well, since we have this fragment "it's basically the radio-labeled piece" "of DNA and since we see the radio-labeled DNA" "it means that it had bound." "It was bound to this Gene A" "which means that Gene A was in this cup of DNA."