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..
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