I'm gonna show you how to do the freeze and squeeze gel extraction method. The
freezing and squeeze gel extraction method is a really fast way to get your DNA out
of an agarose gel and ready for a ligation. The principle of the freeze and
squeeze method is that once agarose gel has been frozen and centrifugation
force is applied to it, the liquid and the DNA will come out of the gel leaving
the agarose behind. So to do this we need to make a filter cup to catch
the agarose during the centrifugation process and separate it from the aqueous
DNA phase. It's important if you use this method for setting up the ligation
directly, that you use TAE buffer in your gel because TAE buffer is compatible
with most molecular biology enzymes like ligase. TBE buffer is not compatible
with that.
So in order to make this, you could buy
the filter cups already from
from bio-rad,
or you can make them for next to nothing with a filter pipette tip and a
standard Eppendorf tube. I'm going to make two different sizes here. We have our
filter tip 10 micro litre pipette tips and we have our filter tip 200
microlitre pipette tips, the 200 microlitre pipette tip will give us a
bigger reservoir so if we have a bigger a bigger piece of gel, we'll be able to
put that put that piece of gel in there. If we have a very very precisely cut
band out of a gel, it can be desirable to use the smaller tip because we'll get a
little bit better volume yield out of using that smaller tip. So
here what we need to do is we need to cut this, this is already cut; we need
to cut this so that it can actually fit in the tip, I mean in the tube
and I don't have to be ultra precisely accurate right so it fits in there and
with that way we can still close this tube.
Same thing for the 200 microlitre tip we need to
cut this so they can't fit in here
the reason we'll get a little bit less yield with this is that you'll often
have some droplet in the bottom of this filter tip at the end it's hard to get
out. So it's desirable to use the small one
if you can. Okay I have two different pieces of agarose gel that I've already
cut on the transilluminator, this small piece I'm going to put into our small
reservoir cup
here.
It doesn't have to go all the way in, it just has to be in
there. I'm going to close this.
We have this bigger piece of gel here that wouldn't fit into the smaller
reservoir cup, the 10 micro litre pipette tip, so we'll put that into the 200
microlitre pipette tip. Again it doesn't have to go all the way down in there.
Close that up.
So I'm gonna put this in -80, I already have a rack that
stays in the - 80 freezer and is already cold. Put that in there and put that in there for
five minutes.
Five minutes is up.
I'm gonna take this whole rack over to the centrifuge quickly, we cannot allow
it to thaw out before it spins for three minutes at maximum speed. Three minutes is up.
Let's take one out, to see each one at a time. If you look here closely you can
see that the agarose stayed on top of the filter and all the liquid with the
DNA is on the bottom. Look at the bigger filter tip and see a big chunk of
dried up agarose right there on top of the filter and a bunch of liquid here.
You'll notice some with this one there's a lot of liquid outside of the tip but a
whole lot of it inside of the tip too. This is this is the undesirable part of
using the bigger reservoir, so to get these out we use a pipetter for the
the 10 micro litre pipette tip we'll use the P 20.
We'll just put it in here gently and if you have any let anything left over,
you see actually there is no droplet inside of the tip all of the liquid is
left behind.
For the p200 tip we have to use the P 200 get that out
and what you're going to want to do is you see there's a lot of the DNA, the DNA
and liquid in the tip so you're gonna want to blow that out and hopefully you
got most everything out, just a little bit of a droplet on the outside
of that tip. I'm not gonna worry about that.
Of course the bigger gel fragment resulted in more volume obviously. An
important consideration is basically the more precisely you cut your band out, the
higher the DNA concentration will be in in that tube and since you often set a
ligation of just a total of five microliters and maybe only use half a
microliter to one microliter in there, it's desirable to cut your gel very
precise so that this DNA is ready to go into a ligation. If you wanted to further
purify the DNA and get rid of the TAE buffer and the ethidium bromide and things
like that, you could phenol:chloroform extract this DNA and then you would have
ultra pure DNA, however, I've cloned lots of genes just directly by using that
liquid. Thank you for watching!