We're now going to perform agarose gel electrophoresis.
Electrophoresis is a technique used to separate molecules based on their charge and size.
An electric current is run past through a gel, and the samples move
along the gel depending on their charge.
Okay, we're going to do agarose gel electrophoresis to check our PCR sample.
This will allow us to check that the amplification has worked
and we will put our sample in the agarose gel, we will run the electrophoresis
and we will be able to tell if there is a band present,
and this will let us know that the DNA amplification has been successful.
To run a DNA agarose gel electrophoresis you first have to prepare the gel,
and that is done using a gel former, and the ends of the gel former are taped up.
Three pieces of tape are used.
When the gel is initially prepared,
it's in a liquid form and it will set in this tank
so both ends of the gel form are taped
and pressure is applied to ensure there are no gaps where the gel can actually
leak out of the former.
This is done at both ends of the gel forming tank.
So the gel former is now ready to have agarose gel added.
The --- is present to produce sixteen wells in the gel
and this is taped into position.
You can see the gel former has grooves along the length of the gel former.
This means you can add another comb of sixteen wells
if you have more than sixteen samples to prepare, like so.
Okay, so we'll now prepare the agarose gel.
For preparation of the gel we require hundred mills of Tris-Borate-EDTA
which is measured out using a measuring cylinder.
So we have a hundred mills of our Tris-Borate-EDTA
and we are going to prepare a one percent solution of gel.
So we need to add one gram of the gel to the Tris-Borate-EDTA.
One gram of agarose gel has been weighed into the conical flask
and to this is added the hundred mills of the Tris-Borate-EDTA.
This is mixed, and this solution will be heated to dissolve and melt the agarose
and then it will be poured into the gel former.
The agarose gel has now been dissolved and to the gel will be added
three microliters of ethidium bromide.
Ethidium Bromide is a compound which will attach the DNA,
and this will enable us to visualize the DNA under UV light.
So the ethidium bromide is well mixed into the gel,
and the gel is poured into the gel preparer.
You then ensure there are no bubbles present on the surface of the gel.
You can use a pippet tip to get rid of any bubbles which are there.
Now we have to wait until the gel sets and then it will be ready for us to add our samples.
Once the gel has set, the tape is removed from the ends of the preparation.
The comb is removed, leaving the sample wells, and the gel is placed in the electrophoresis tank.
Now three hundred mills of Tris-Borate-EDTA buffer is poured into the tank
to cover the gel to the buffer ethidium bromide has been added
and the buffer is added to the maximum fill line on the electrophoresis tank.
The gel is now ready to have the samples added,
so they prepared the set to the correct volume for each sample.
A fresh tip is used for each sample,
so the first sample is taken and carefully dispensed into the well.
Care should be taken not to put the tip through the bottom of the gel
when you're adding your sample to the electrophoresis gel
and a fresh tip used for each sample.
The sample settles at the bottom of each well,
ensuring that the DNA travels through the gel and not across the top of the gel.
Once the samples have been added,
the cover is put on the electrophoresis tank and the wires are connected to the electricity supply.
The current is then switched on,
that is adjusted to the correct voltage,
and now the gel is left to run for thirty minutes.
Once the gel has run its course, the power supply is switched off,
the lid is removed from the tank, and the gel is removed,
and the excess buffer is allowed to drain off and then it is placed on some tissue paper.
The gel is now ready to be read in the UV transilluminator.
The gel is placed in the illuminator.
The door is closed
and then the gel is focused upon.
So here we have an image of the gel focused and the bands are fluorescing in the UV light.
On the left here, we have the reference ladder with known DNA sizes
from ten kilo base pairs down to a hundred base pairs here,
and you can see that the smaller base pairs move the furthest.
And of these six samples, which are loaded, three are showing bandings.
These three have bands just over two hundred base pairs,
and the one in the middle has one about hundred and fifty base pairs as well.
So that would suggest these two are homozygous whereas this one is heterozygous,
so it has one gene, which is this size, and another piece of DNA, which is this size.
So once we visualize the gel, we can print a picture of it
and keep that as a permanent record of our analysis.