- Separations and purifications questions
- Simple and fractional distillations
- Principles of chromatography
- Basics of chromatography
- Thin layer chromatography (TLC)
- Calculating retention factors for TLC
- Column chromatography
- Gas chromatography
- Gel electrophoresis
- Resolution of enantiomers
Learn how gel electrophoresis separates DNA and protein fragments based on size and why one would use agarose gel electrophoresis versus SDS-PAGE. By Angela Guerrero.. Created by Angela Guerrero.
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- I'm very confused, I understand that DNA travels towards the positive end but shouldn't the positive end be the cathode? The cathode is positive in a galvanic cell...or is this instance different?(15 votes)
- I believe it's like this because this is an electrolytic cell. In an electrolytic cell the charges are swapped. You might be confusing it with a galvanic cell. I was confused too initially :)(18 votes)
- this might be a silly question but here it goes: I know the movement of fragments are substantiated by the forces applied by the electrical field, and the heavier the fragment, the slower it moves. However, is it possible that the fragments might carry different charges and thus the forces applied might vary, and then that will affect the kinetics?(6 votes)
- Yes the charge does affect the kinetics. In terms of DNA gel electrphoresis, DNA molecs already have a constant mass to charge ratio because every nucleotide has a -ve phosphate group. However for proteins (made up of different amino acids with varying charge) the charge can affect the kinetics. This is why the proteins are denatured with SDS which coats the proteins with -ve charges proportional to the mass, therefore all the protein fragments have a constant mass to charge ratio. This way the protein will only be separated by mass and not charge difference.(14 votes)
- What does it mean for the green sample to have two fragments of different sizes? Shouldn't each well have only one band, because the DNA in that sample should all be the same and have the same size? Why would there be one fragment larger than another?(7 votes)
- This is based on where the restriction enzyme cuts the DNA.If it recognizes only one site it will cut the DNA into 2 fragmments .(11 votes)
- By any chance, do you know the difference between Native PAGE and SDS-PAGE for protein electrophoresis?(6 votes)
- What do the bands that match represent? And what does the ladder go up by?(2 votes)
- The bands that match represent fragments of DNA that are of similar size. This doesn't necessarily mean, however, that the DNA of those matching bands are the same.
The ladder doesn't go up by a set number, as there are different DNA ladders that can be used to standardize size, and the bands are not equal distance apart. For example, a commonly used standard is the 1 kb ladder, which bands go 100 bp, 200, 300, 400, 500 then it jumps to 650 bp. Thus, the information regarding the size of the ladder's fragments will be given by the company who makes the standards.(2 votes)
- At1:06, the cathode and anode are labeled differently than a gel box would look like in the laboratory. The anode should be labeled red, and the cathode should be black. The way to remember is that DNA "runs" to Red. I don't know if the colors were intentional but this could be a source of confusion. Also, around2:56she mentions what a gel would "look like." First off, the only thing visible to the naked eye would be the loading dye, which usually has three distinct colors that should separate and encompass the smallest fragments, the middle of the run, and the largest fragments, to give a visual cue as to where the DNA fragments are. So essentially, all of the samples loaded, even the ladder, would look identical to the naked eye (even if there was a well with no DNA and only loading dye [perhaps PCR failed]). Secondly, in order to view the DNA, both the gel and the buffer need to have a dye (different from the loading dye as EtBr is not visible to the naked eye at the small concentrations needed for this purpose [highly concentrated, it is a deep yellow/orange color]), such as ethidium bromide (Which is denoted as EtBr [not to be mistaken for bromoethane]) that will intercalate with the DNA and fluoresce orange with exposure to UV light.(2 votes)
- What do you mean by "both the DNA and the buffer need to have a dye"? I've been working on some EtBr gels and we only use dye for the loading buffer of the DNA and ladder, so that we can see their approximate progression through the gel. All of the real visualization is done via UV.(2 votes)
- Hi, is this considered a chromatographic method?(2 votes)
- not exactly because chromatography is a different process
"Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction"
it is an electrochromatographic method though.
"Electrochromatography is a chemical separation technique in analytical chemistry, biochemistry and molecular biology used to resolve and separate mostly large biomolecules such as proteins. It is a combination of size exclusion chromatography (gel filtration chromatography) and gel electrophoresis"
Electrochromatography is more chemical and electrical while chromatography is just physical.
Today, we'll be talking about gel electrophoresis. What is gel electrophoresis, you might ask. Well, it's a lab technique usually used in the biochemistry lab for separating out DNA or proteins based on their size. And let's talk about how it works. So first, you need to have the gel. This can be made out of different kinds of substances, such as agarose and polyacrylamide, both of which I'll discuss later. And the electrophoresis part of it means that you need to have an electrical field passing through the gel to get the bands to move. So to create an electrical field, we have to have a cathode and an anode. The cathode is on this side. Remember from general chemistry that at the cathode, reduction takes place, so you'll have a negative charge. But on the other side, where the anode is, you'll have a positive charge, because this is where oxidation is taking place. And to power this, you'll need some kind of battery to connect them. And these are both usually also connected to a big box that contains the gel electrophoresis apparatus. In order for charge to flow across, you need something that will conduct electricity, so you have a buffer that's usually composed of different ions. So this is completely covering this entire gel. And remember that it's always there, but I'm going to erase it just because it's going to get a little messy for what I need to show you next. So next, you'll load a sample of DNA. In order to load your sample, you'll need to take a pipette and put it into one if these wells. So usually you'll want to make sure that you have a dye mixed in with your sample so that you can see it as it's running. So I'll show that as a pink line here, so I've filled that well, followed by yellow here, and green here. So that's just the color of the dye. It can really be any color you want. This will just help you track the movement of the bands later on. Once you have these bands, what will happen once you turn on the electrical field? Remember that DNA has a negatively charged backbone because of all the phosphate groups, so what you'll actually observe is that they'll travel towards the positive end. So maybe after a short period of time, what you'll see is that-- it'll look something like this. Maybe the pink will have split up into a few bands, indicating that there's a few different sized fragments in there. With the yellow, you might only see one band. And with the green, you might actually have two bands, but they're so close together still that it's kind of hard to tell. So how can you really see what's going on? The solution is to let this run for a longer period of time. And eventually, what you'll see is shown here at the bottom. You'll see that whatever sized fragments were in your original samples have effectively split up by their size. And note that in pink, you'll always want to load something known as a DNA ladder. A DNA ladder is like a standard. This is something that you buy from a company, and they tell you exactly what sizes their fragments so that you can match them up to your unknown samples. So this could be 400 base pairs, 200 base pairs, and 100 base pairs. And as you'll note, the smallest fragments travel the furthest. This is because the smallest things are really easy to push with the electrical field. But when you have such a big molecule, or rather a big DNA fragment, it can be hard to move. So you can see that the 400 base pair doesn't move too fast or too far. And what's this tell us about our unknown yellow and green samples? This shows us that the yellow sample has a band that's 200 base pairs long. And the green sample actually was composed of two different fragments, one that was 100 base pairs and another that was 200 base pairs. So what would we do with this information? If you needed a particular size of DNA, say for the next step of your experiment, if you wanted to insert it into a plasmid or a vector, you could cut this out of the gel and use it for that. Now let's talk about the two kinds of gels that are most commonly used. The first is agarose, and the second is SDS-PAGE. So agarose is a gel that's usually used for separating big pieces of DNA. So if you think about the pore size in the agarose, it has pretty big pores, so imagine it looking kind of like this. The gel is pretty big. There's big holes here, so that you'll be able to separate out the big pieces of DNA that come through. However, if you're trying to separate out little pieces, it won't be that obvious, because they'll all just race through these giant holes. So remember that this is for big DNA fragments. Usually, this is for DNA that's bigger than 50 base pairs. SDS-PAGE, on the other hand, can be used for very small things. So imagine that being a much finer weaving with smaller pores. Although this can be used for small pieces of DNA, it can also be used for proteins. You might be wondering, what does SDS-PAGE even stand for. The SDS part is Sodium Dodecyl Sulfate. This is a chemical agent that denatures proteins, disrupting any non-covalent interactions they may have. This makes it so that the charge of the proteins isn't a factor when they're separating out onto the gel, and they're only being separated strictly by size. The PAGE part is PolyAcrylamide Gel Electrophoresis, or we'll just leave it at GE. So polyacrylamide is the substance that gel's made out. So how can we remember the difference between these two types of gels? Remember that SDS-PAGE is for small DNA or protein cells. S for small, and S for SDS. And agarose is for bigger fragments of DNA. So today we've talked about how you would setup a gel electrophoresis, why it works, and how you would want to pick the substance but your gel's made of. If you were really doing this in the lab, now that you have your fragments of known size of DNA or protein, you could either sequence them or use them in other molecular techniques.