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- Gel electrophoresis
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Introduction to gel electrophoresis. How it's used to separate DNA fragments or other macromolecules.
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- Does the DNA migrate towards the positive electrode because it is negatively charged? What about the other macromolecules you spoke about? Would you set up positively charged molecules on the opposite end?(9 votes)
- Yes it migrates towards the positively charged anode because of the net negative charge on the DNA. You can also use SDS which is a soap that can denature DNA, RNA, or protein to give them a net negative charge and a constant charge to mass ratio, which will allow them to migrate in the presence of an electric field. They will also only migrate on the basis of charge which is what you want (SDS coats the sample proportional to their mass). But the side effect to using SDS is that it denatures the sample.(17 votes)
- How can you be sure which of the vials you are seeing? Do they ever veer into #3's space?(7 votes)
- You put the liquid carefully in these wells, indents in the gel, and they move through the gel straight ahead towards the anode (positive end) so you get quite straight lines without having to worry about them veering out of their "lane".
Of course, you can make mistakes that might cause contamination of wells. Since you are filling the wells submerged (placing it in the liquid) you could accidentally "miss" the well spraying out the content in the general liquid, but from what I've seen the liquid is always colored so you see quite easily if you've done that, and I expect that if you do the electrophoresis then it'd quite easily and quickly wander toward the anode (since there won't be much resistance in the liquid, compared to the gel) and wouldn't necessarily influence your other answer. Another issue could be if you poked a hole through the gel and into another well, that could mix it and cause problems.
But generally it's not a problem(11 votes)
- Can you use this technique in a crime scene to figure out if the DNA that you collected from pieces of evidence matches the original DNA? Also, can you really use this to put DNA strands in order from tallest to shortest?(6 votes)
- You can really use this to put DNA strands in order from tallest to shortest. I don't think it would really help you with a crime scene because most human DNA would separate in generally the same way with gel electrophoresis. It doesn't distinguish differences in DNA, but gives you lengths.
That said, if DNA you found on a crime scene matches the DNA you were examining exactly, I'm sure you'd be quite suspicious and likely to run more tests.(8 votes)
- If I keeps the gel running for too long, the shorter bp of DNA will sure migrate towards the end of gel, then I wouldn't really know the amount of bp after that. Is there a standard time for running of gel?(7 votes)
- Even if the shorter bp of DNA migrates farther than it is supposed to, the other channels of DNA strands will also have DNA fragments going farther, so it can still be used as a reference. There is not a standard time, but naturally, one should be careful not to let it run too long, so all of the DNA does not go to one end of the gel.(1 vote)
- Why would you want to know the length of your fragment?(5 votes)
- To separate fragments of DNA, RNA or proteins - based on their molecular length.
Electrophoresis is used to extract certain fragment. How could you do that if all same size? In that case all would travel same speed and you will not be able to tell them apart.(4 votes)
- Is the difference in distances linear? If not, how do we measure relative distances?(3 votes)
- The standard way to graph this relationship is log(length of nucleic acid molecule) vs. distance.
However, in my experience it is actually very uncommon to make a standard curve — when doing research a rough estimate by "eye" is generally good enough.
Agarose gel electrophoresis has relatively low resolution and for various reasons the bands often run somewhat differently in different positions across the gel — if you need to know the exact size of a DNA fragment you would probably just sequence it!(4 votes)
- How would you know how long to wait before stopping the electric current? Sal said at5:32that the DNA could potentially fall off the edge if you waited too long. So wouldn't that affect the position of the sample DNA relative to the ladder, and therefore your conclusions?(3 votes)
- there are lab protocols that know how long to keep the machine running. from experiments they would determine what is an appropriate and standard time and current to use. relative to that all the measurements would then be analyzed
source to consider: https://www.youtube.com/watch?v=vq759wKCCUQ(3 votes)
- How does finding the length of the specific DNA molecules help one conducting genetic engineering? I'm currently studying this in my Biology class and I don't fully understand it. What I mean by this, is why would you need to know the lengths of the DNA molecules?(3 votes)
- Sometimes you do not use Gel electrophoresis to know the lengths but just to separate fragments. Usually, gel electrophoresis is used after PCR to separate target sequence from the rest we would like to use in genetic engineering.(3 votes)
- But how do you know, how long you are supposed to wait until you have to turn off the electricity? I mean, at5:29Sal says that if you wait for too long these strands of DNA could fall of the other edge. How do we know when it's the right time, that these strands haven't gone too far or opposite?(2 votes)
- The samples are mixed with one or more negatively charged dyes that migrate at a similar rate to DNA molecules of a certain size. For example, a very commonly used dye is bromophenol blue, which migrates at the same rate as ~350 bp DNA molecules§. This allows you to estimate how far your sample has run and stop the gel when you expect to have achieved good separation of the band(s) you are interested in. The migration of the dye also allows you to verify that you are running the gel in the right direction.
You can then use UV light to visualize the bands in your ladder and samples — if you haven't run them quite far enough you can just put the gel back in the gel box and continue running it.
§Note: The migration rate also depends on the percentage agarose in your gel. You can see a chart describing many loading dyes here:
And read more about this here:
- If I was to set up this gel electrophoresis (hypothetically) and made the gel huge in length, and the anode and cathode strong enough to allow this to work, and enough buffer and so on. Would there become a point where the fragments are seperated so much that individual bases could be able to seen and plotted.(3 votes)
- Good question, but the answer depends on what kind of gel you are asking about.
(I'm assuming that you are interested in separating molecules that differ in length by a single bp, rather than separating individuals bases.)
For the type of gel (agarose) being discussed in this video — the answer is no. The primary reason is that two processes are happening while the gel runs. First, the DNA molecules are wiggling through the pores in the gel under the influence of the applied voltage. Second, the DNA molecules are diffusing (i.e. moving randomly in all directions). This results in the bands spreading out over time, which limits the ability to resolve small differences.
If we need high resolution, the traditional solution (e.g. for sequencing gels) was to run vertical polyacrylamide gels — these have much smaller pore sizes and thus can more easily separate DNA molecules differing in length by only a single base pair. IIRC, this can be done on a gel that is <40 cm long.
There are also newer techniques that allow high resolution separation of nucleic acids — one example is capillary electrophoresis.
- [Voiceover] 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? In vial number one. How long are those fragments? How many base pairs? How long are they? Well, you might say, well why don't I just take them out and count them? Except for the fact that they're 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 5000 base pairs, well that's going to be approximately one to two micrometers long if you were to completely stretch it out. And we can't even start to think about how thin the actual diameter is, if we just, but length-wise, the long way, it's only going to be one to two micrometers which is super duper small. This is one to two thousandths of a millimeter. So that's not going to help us to somehow try to manipulate it physically with our hands or with, 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, it could be used for RNA, if 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. And it's called gel electrophoresis because it involves a gel, 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 gel, that's inside of a, that's embedded in a buffer solution. So this gel, the most typical one is agarose gel, that's a polysaccharide 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 gonna 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 can view these wells as little divets 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 of this buffer, so you can see the buffer I drew, this fluid, and that's really just water with some salt 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 in the basic or acidic side, it might actually affect the DNA, or affect the charge on the DNA. Now what we're going to do is, we're gonna put a charge across this whole setup. Where the side where the wells are, where we're gonna place the DNA, that's going to be where we're gonna put the negative electrode, so that's our negative electrode there. And the other end is going to be our positive electrode. And we're going to use the fact that DNA has a negative charge at the typical pHs, or the pHs that we are going to be dealing with. Now we can 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 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 gonna be the same regardless of length. And so what determines how far something gets, how much it migrates over a certain amount of time, is how small it is. Remember, we have this 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 mesh, this net, this sieve, well smaller things are gonna 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 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 and 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 come back and you were to see this migration, you were to see this migration occur, and the longer you wait, the further these things are gonna get. In fact, if you wait too long they're gonna fall off all the way over the other edge. Is, if you just saw this you'd say okay, well 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 there are some really short strands, maybe there were some really short strands in that, what I'm drawing as, that orange group right over here. So, what I just did right over here 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. And so let's say you go get the DNA ladder, I'm gonna draw it in pink, so you literally could buy this. You can buy it online. And the standard solution let's say it separates like this. So it separates, 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 whichever one you choose to buy, that this grouping here, this 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, how many base pairs these are. So you say okay, 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 green one here, well it's a little bit longer than 1500 base pairs, it didn't migrate quite as fair as this big bundle of 1500 base pairs guy 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're actually going to look for. Now the other thing to appreciate is, when you see, when you see 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 DNAs that you're looking at. And this is, they're not all stretched out like that. Remember, even something that is 5000 base pairs long is only going to be one to two micrometers if you stretch it out. So, you wouldn't even be able to see it, it's a thousandth of a millimeter. You wouldn't you even be able to see it. So this is many, many, many molecules of DNA, is migrating that far. And they wouldn't even 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 they're 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 it ethidium bromide. And ethidium bromide is called an intercalating agent, and it's a molecule, you can see the ethidium 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 ethidium that has fit itself, that's why we call it intercalating, it has 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 ethidium bromide into all of your DNA right over here, and then as it migrates, and then if you were to turn on 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, 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 5000 base pairs, this right over here is our 1500 base pairs, and this right over here is our 500 base pairs. And then let's say you had some solution of some other DNA, and you wait a little while, and you see look, it migrated not quite as far as a 500 base pair, so it must be 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. Now once again, 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 read-outs from gel electrophoresis. So now you know what's actually going on here. This isn't a strand of DNA, this is a big, this is a bunch of DNA that has been tagged with some type of a dye, or the ethidium 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.