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Video transcript

let's say that you have some vials here and you know that in the solution you have fragments of DNA in each of these and what you're curious about well what about the DNA fragments in our in this first vial and vial number one how long are those fragments how many base pairs long are they well you might say well one I just take them out and count them except for the fact that they are incredibly small and incredibly hard to handle even a fairly large fragment of DNA let's say we're talking about something that's on the order of five thousand five thousand base pairs well that's going to be approximately one to two micrometers long if you were to completely stretch it out and and we can't even start to think about how thin the the actual diameter is if we just butt lengthwise the long way it's only going to be one to two micrometers which is SuperDuper small this is one to two thousandth of a millimeter so that's not going to help us to somehow try to manipulate it physically with our with our hands or with the you know kind of rough tools so how do we do that and we could have other vials there how do we see how long the DNA strands that are sitting in those vials actually are and the technique we're going to use gel electrophoresis it actually could be used for DNA strands khabees for RNA could also be used for proteins any of these macromolecules to see how long are those fragments and so let me write this down gel electrophoresis electro for rhesus and it's called gel electrophoresis because it involves a gel it involves it involves electric charge and phoresis is just referring to the fact that we are going to cause the DNA fragments to migrate through a gel because of the charge so phoresis is referring to the migration or the movement of the actual DNA so how do we do this well here is our set up right over here we have our our gel that's inside of a but that's that's embedded in a in a buffer solution so this gel the most typical one is agarose gel that's a poly that we get from seaweed and it's literally a gel it's a gelatinous material and what we're going to do is is we're going to put we're going to take samples so we might take a little sample from this one right over here and we'll put it in this well right over here and you could view these wells there's little divots in the gel you could take a little sample from here and put it into this well and then you could put a sample from here and you could put it in that well and it's going to be bathed inside this buffer so you can see the buffer I drew this fluid and that's really just water with some salts in it and the buffer is going to keep the pH from going too far out of bounds as we place a charge across this entire thing because if the pH gets too far and the basic or acidic side it might actually affect the DNA or affect the charge on the DNA and what we're going to do is we're going to put a charge across this whole this whole set up where the side where the wells are we're going to place the DNA that's going to be where we're gonna put the negative the negative electrode so that's our negative electrode there and the other end is going to be our is going to be our positive electrode and we're going to let use the fact that DNA is has a negative charge at the typical PHS or Inlet the pH is that we are going to be dealing with now we could go back into previous videos and we can see it right over here you see these negative charges on our phosphate backbone and so what is going to happen what is going to happen once we once we connect both of these to a power source and then this side is negative and this side is positive well the DNA is going to want to migrate now let's think about what will happen will shorter things migrate further or will longer things migrate further well you might say well longer things are going to have more negative charge so maybe they go farther away but then you also have to remember that they're also moving more mass so their charge per mass is going to be the same regardless of length and so what determines how far something gets how much how much it migrates over a certain amount of time is how small it is remember we have this agar agarose gel and people are still studying the exact mechanism of how this DNA or these macromolecules actually migrate through the polysaccharide but if you imagine this polysaccharide is kind of this this mesh this net deceive well smaller things are going to be able to go through the gaps easier than the larger things and so if you let some time pass if you let some time pass some of the DNA let's say this DNA gets around there let's say and I'm just color you actually wouldn't you see these colors let's say this DNA gets around that far so it doesn't get as far let's say that this DNA doesn't migrate let's say it has some that migrates that far let's say it has some that migrates that far and so if you just saw this you wait some amount of time and you were to come back and you were to see this this migration you were to see this migration occur and the longer you wait the longer the my the further these things are going to get in fact if you wait too long they're going to fall off all the way over the other edge is if you just saw this you'd say okay well this this strand right over here these must be smaller DNA molecules they must be shorter these must be a little bit longer and these must be even longer than that and this grouping right over here is going to be the longest of all so this was a mixture of some longer strands and still longer ones but not quite as long and for example maybe maybe there are some really short strands maybe there were some really short strands in that what I'm drawing as what I'm drawing as that orange group right over here so what I just did right over here this will tell this would this could tell you the relative length of these strands but how would you actually measure them well that's where you can go find standardized solutions which we call a DNA ladder so let's say you go get the DNA ladder I'm gonna draw it in pink so you literally could buy this you could even buy it online and the standard solution let's say it separates like this so it separates like that goes there let's say some of it goes like there and some of it goes like there well you would be able to know from the labeling or what whichever one you choose to buy that this grouping here these this is all of the DNA that is 5000 base pairs let's say let's say this right over here is 1500 base pairs and let's say this over here is let's say this over here is 500 base pairs long and so now you can use this DNA ladder these standardized ones to gauge how long the D how many base pairs these are so you say okay this is this blue one here this is a bunch of DNA that's a little bit longer than 500 base pairs but it's shorter than 1500 base pairs you can see this this green one here well it's a little bit longer than 1500 base pairs it didn't migrate quite as far as this big bundle of 1500 base pairs that did and so then you can get a better approximation and you can choose your ladder based on what you think you are going to find there what you are actually going what you're actually going to look for now the other thing to appreciate is when you see when you see the the DNA having migrated this far you might say okay is this one DNA strand is that one DNA strand that I'm looking at and just going back to the measurements no that is many many many many DNA's that you're looking at and this is they're not all stretched out like that remember even something that is five thousand base pairs long is going it's only going to be one to two micrometers if you stretch it out so it wouldn't even you'll see that it's a thousandth of a millimeter you wouldn't even be able to see it so this is many many many mountain molecules of DNA is migrating that far and they would have to be that small to be able to migrate through that polysaccharide gel now the last thing you're probably saying is okay wait but how am I even seeing it over here how do I actually see this DNA especially if there are these super super small molecules and the answer is you put some type of marker on the DNA that will make them visible some type of dye or something that might become fluorescent and one of the typical things that people often use is Atheneum bromide and Atheneum bromide is called an intercalating agent and it's a molecule you could see the Atheneum right over here these are two DNA two backbones of DNA you can see the base pairs bonding here and then this right over here that is at idiom that his fit itself that's what that's why we called intercalating it his fit itself in between the rungs of the ladder and when it does so inside of DNA it actually becomes fluorescent when you apply UV light to it so if you put this atheneum bromide into all of your DNA right over here and then as it migrates and then if you were taunt around in a UV light it would become fluorescent and you would actually see these things and so if you wanted to see what it actually would look like in real life well this is what it would look like when you were to if you were to look at it straight on where this would have been a well though let me make it a little bit easier to read so right over here would have been the well where you would put the DNA ladder and it would come up with standardized measurements maybe that's our five thousand base pairs this right over here is our fifteen hundred base pairs and this right over here is our five hundred base pairs and then let's say you get some solution of some other DNA and you wait a little while and you see look it migrated not quite as far as the 500 base pair so it must be a little bit this must be a bundle of things a little bit longer than 500 base pairs but for sure a lot shorter than 1500 base pairs and once again it doesn't have to have just one fragment length you could have had another group that was maybe right at 1500 base pairs and you've probably seen this whenever you see people talking about genetic analysis and things like this you're often seeing people look at one of these readouts from gel electrophoresis so now you know what's actually going on here this isn't a strand of DNA this is a big there's a bunch of DNA that has been tagged with some type of a dye or the Atheneum bromide idiom bromide or something like that and it's a bunch of those molecules and they've migrated based on the charge they're trying to get away from that negative charge to the positive charge and the smaller molecules this is a bunch of small molecules right over here are able to get further because they're able to get through the mesh of the agarose gel
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