Today we are going to explore the concept of electrophoresis. So what
exactly does electrophoresis mean? Electrophoresis or electro meaning
"electric field" and phoresis meaning "migration" is the method of using an
electric field to separate molecules based on their size and polarity. Here we
will be utilizing gel electrophoresis as a means to separate samples of DNA
molecules. DNA is a negatively charged molecule that can be cleaved into
different sized fragments using molecular scissors called restriction
enzymes. In this demonstration we'll be separating for DNA samples each
containing millions of individual fragments. These fragments can be one or
more different sizes of nucleotide base pair lengths sample a contains DNA
fragments of the same size each fragment is two thousand base pairs sample B also
contains DNA fragments of the same size each fragment is 1,000 base pairs sample
C contains DNA fragments of two different sizes one has 300 base pairs
and the other 500 base pairs sample D contains DNA fragments of all four sizes
with the samples ready it's time to start setting up the gel electrophoresis
chamber in this demonstration we'll be working with the mini one
electrophoresis chamber we will begin by plugging in the unit
with the lid removed a precast agarose gel is placed into the tank agarose is
used as a medium to separate molecules because of its porous spongy matrix
next 135 milliliters of running buffer is added to the tank by pouring half of
the buffer into one side of the chamber until the air trapped under the tray is
pushed out the remaining buffer should then be poured into the other side of
the chamber until the gel is fully submerged the buffer is full of
dissolved solutes it's conductivity creates a uniform distribution of ions
from one electrode to the other through the gel electrophoresis chamber as the
electric charge flows through the agarose gel it will mobilise the DNA
samples causing them to move since opposites attract and the DNA is
negatively charged it will begin to migrate toward the positive electrode
at this point the low intensity blue light can be switched on this will make
it easier to see the wells in the agarose gel to load our DNA samples
wells are cup-like depression x' that are created by use of a comb when the
gel is cast and are used as the location for which to place the samples for
separation the number of wells a gel has can vary based on the comb selected as
you can see our gel has 6 wells because we have just four samples we'll only be
using four of those wells using a micropipette the DNA samples are
carefully added to the wells in the agarose gel during this step be very
careful when inserting the tip of the pipette it should only be lowered about
halfway into the well to avoid penetrating the bottom be sure to record
the order in which the DNA samples are loaded it is also very important to
change the pipette tips between each sample to avoid cross-contamination
with the gel loaded and the lid replaced the unit is ready to be switched on
as the current is applied it flows from the negative electrode to the positive
electrode since DNA is negatively charged it migrates toward the positive
electrode through the agarose loading and tracking dyes have been added to the
samples this dye makes it easier to load the samples since DNA is virtually
colorless while also creating a dye front ahead of the migrating DNA on the
molecular level the matrix of an agarose gel looks a lot like a sponge full of
pores as the DNA molecules migrate through the gel they will encounter
obstacles and bump into structures we can imagine the agarose matrix acting
like a series of barriers that the DNA has to pass through the length of the
DNA fragments has a direct correlation to the speed at which they are able to
navigate the agarose this means that the smaller the molecule the less it will
collide with the agarose allowing it to move through the gel more quickly by
comparison the larger the molecule the more often it will collide with the
agarose causing it to move more slowly through the gel as a result the smaller
molecules slip through the agarose faster than the larger molecules as the
molecule bands move through the gel they are led by the tracking dye this dye
front migrates the fastest through the agarose and serves as a visual indicator
for when to stop the electrophoresis process once the dye front reaches the
end of the gel the power is cut off the agarose gel is now ready to be
examined and the DNA banding patterns analyzed each of the individual bands
represent millions of pieces of DNA of similar size that are stuck within that
section of the agarose compare the loaded samples to the
banding pattern for analysis
the analysis of DNA banding patterns like those demonstrated in this video
can be used for a variety of applications including forensic science
food safety paternity testing and much more special thanks to mini one systems
for their support in making this video possible