A technique that I have used several times both in research and my
biology classes is gel electrophoresis[1].
Gel electrophoresis can be used to separate DNA, RNA and proteins by length. An
agarose gel is made up of agarose, 1x TAE (Tris-acetate-EDTA), and Ethidium
Bromide. It is then poured into a tray that is placed in a holder that has
sides that hold the agarose in the tray until it has hardened. A comb is also
placed in the gel in order to create wells for loading samples. The agarose gel
that is used as a separation medium has a web-like structure which allows
smaller particles to pass through more easily than larger particles. This gel
is then placed in a small tank and covered in a solution containing salts. This
solution conducts electricity which carries the molecules through the gel. Samples
are then loaded into the gel using a pipette. Because the samples are
colorless, they are mixed with a dye before being loaded into the gel. This
makes loading easier and allows one to see the movement of the samples. A
ladder is also loaded along with the samples.
The tank that the gel is placed in has a positive pole and a negative
pole. DNA has an overall negative charge because of the negatively charged
oxygen found in the phosphate groups in the backbone of the DNA. Because the
DNA has a negative charge, it moves toward the positive pole of the tank when
the electrophoresis unit is turned on.
After a specified amount of time, the electrophoresis machine is turned
off and the gel can be looked at using ultraviolet light. A small amount of
Ethidium Bromide is added to the agarose gel. The Ethidium Bromide attaches to
the DNA and then glows when exposed to UV light. This allows one to see where
the DNA ends up after running the electrophoresis. The location of the bands
can then be analyzed by comparing them to “markers” that are found in the
ladder. These “markers” are segments of DNA of known sizes.
In doing this technique several times there are a few things that I’ve
learned or have been told are important. When loading the gel I usually rest my
elbows on the table and hold the pipette with both hands. I then place the
pipette tip just above the well, taking care not to put the tip too far into
the well (this could cause holes and the sample to spill), and insert the
sample. I’ve learned that this takes practice and I’m still learning. The gel
is very slippery and will easily come off the try if it isn’t held onto. When
lifting the tray out of the tank, I usually grip it in a way that allows me to
have fingers on both ends to prevent the gel from sliding off. I have also
learned that the gel can be wrapped in plastic wrap and stored in the fridge
for another use if there are still available wells.
The following are things that I always double (or sometimes even triple)
check:
·
What I’m adding to the sample. Make sure it’s
dye and not ladder.
·
That the lid is placed correctly. The black
cord/end of the lid is placed on the black end of the tank and the red cord/end
of the lid is placed on the red end of the tank.
·
Make a note of what was placed in each lane.
·
Always run to red! This means that the wells
must be placed at the black end of the tank.
·
The time. One must be careful not to let the
machine run too long because samples can run off the edge of the gel, ruining
all of the work that one already put into a project.
Here's a picture of a gel after it's been run through the gel electrophoresis machine.
[1] "Gel
Electrophoresis" Biology Animation Library :: DNA Learning Center. (n.d.).
DNA Learning Center. Retrieved
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