Gel electrophoresis | Biomolecules | MCAT | Khan Academy

- [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 time of marker

on the DNA, that will make them visible.

Some type of dye, or something that

might become phlorescent.

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
phlorescent 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 phlorescent,

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.