DIY Electrophoresis - a tool for
Here we can seperate a mixture of
molecules using an electric current. Fragments of DNA seperated
to form a "fingerprint" is one application of this technique. A great
project for amateur scientists.
(featured in the New Zealand Science Teacher
journal, No 103, 2003. ISSN 0110-7801)
Soap dish; 9 volt batteries; Aluminium foil
OR nichrome wire; Agarose OR Agar; Baking Soda; Ice-cream container
lid; Glycerol; Droppers or Pasteur pipettes; Test mixture (could be
plant indicator material that is concentrated by evapouration or Human cheek cell DNA); Methylene
Blue (only if DNA was your test mixture)
Dissolve about 1/4 teaspoon Baking Soda in 1 1/4 cups water (3 g
in 300ml water). In the lab you can make a 1% solution by dissolving 1g
in 100ml water.
Agarose is traditionally used but may be expensive. Make a 1% agarose
gel by putting about 1/4 teaspoon of agarose in 1/4 cup buffer
(1g in 100ml). Alternatively use agar (not nutrient agar)
in its place. I have often used a 0.8% agar gel (0.8g in 100ml).
COMB: Use a thin plastic lid similar to an ice-cream
tub lid to cut a "comb" that will rest on the sides of the dish but
have rectangular "teeth" with square ends pointing downward. The 5mm
wide teeth need to be cut to a length that will stop 2 or 3mm from the
bottom of the soap dish. See if you can get four or more teeth on a
ELECTROPHORESIS CHAMBER: Use a
soap dish lid or its base.
- Microwave the agarose or agar gel solution on High
until all has dissolved (this may take about 5 minutes). Allow to cool
to about 50oC (able to be held in hand with out burning). If it gets
too cool and starts to set, simply reheat! You can do this as many
times as required.
- Pour your "hand hot" gel solution into your dish. You
need a gel between 5 and 7mm thick, no more. Immediately place
you comb into the gel about 2 cm from one end. This will form "wells"
in which to place your sample mixtures.
- Leave to set on a level surface.
Carefully remove the comb. We now want to prevent the gel from being
distorted by the bubbles that form at the electrodes. Use a blade
to cut out a 1cm wide strip of gel from opposite ends of the dish where
the electrodes will go. Don't damage the wells that should now be 1cm
from the new edge.
- At each end, place a wide strip of aluminium foil
covering the interior sides to act as electrodes, or alternatively,
place a length of nichrome wire that overlaps one edge of the dish for
attachment of an alligator clip.
- HINT: To speed things up you could pour several gels
and store in the fridge with plastic wrap covering the top. They will
keep for quite a few days.
LOADING THE WELLS:
- Flood the gel with buffer (ice cold sometimes helps)
so that the gel is covered to a depth of about about 3mm.
- Mix 2 drops of glycerol with 6 drops of your sample
on a slide. The glycerol will make your sample more dense so it will
fall into the well rather than difuse into the buffer and be "lost".
Colourless samples can have a drop of Bromphenol Blue added.
- Collect some of your sample in a dropper or pasteur
pipette and place the tip just under the surface of the buffer above a
well. Slowly fill the well. Coloured samples of plant material are easy
- Fill the other wells in a similar manner with the
rest of your test samples. Don't forget to keep a record of what sample
was in which well!
RUNNING THE GEL:
- Use an alligator clip to connect the electrode
closest to the wells to the negative terminal of one of the 9 volt
batteries. Connect 4 other batteries in series by stacking and
connecting oppositely charged terminals in a pyramid fashion. The
remaining positve terminal is connected to the other electrode with
another alligator clip.
- After a while you should see bubbles forming at the
electrodes. Leave the gel until your samples have seperated into
distinct bands.If you had to add Bromophenol Blue wait till it has
almost run to the end of the gel.
- If you have used plant or animal DNA in your sample,
stain the gel with Methylene Blue (0.02 g in 100ml water). Stain for
one hour (at least) then rinse in water.
Record your observations:
Two different samples of food colouring
run at 45 volts for 30 minutes
Points to note:
- NEVER use Ethidium Bromide to stain DNA. It is
- A 1% gel with a slightly alkaline buffer around pH 8
- Sodium chloride buffer works but often causes the gel
to heat up and melt.
- Platinum electrode wire can be obtained from
Crescendo Supplies. We were fortunate when last year they generously
donated some material for these trials.
- Use thin gels with small volume sample wells to get
- Place the comb in the middle of the gel if comparing
other dyes in the lab or other test material. Some bands may migrate to
the positve terminal, others to the negative. You need to allow room
for the differences to be seen!
- Molecules are often electrically charged. A molecule
that has an overall positive charge is attracted to the negative
electrode and vice-versa. DNA has an overall negative charge.
- Small molecules or DNA fragments tend to travel
faster than large molecules or DNA fragments.
- Try using plastic petri dishes as an alternative to
using soap dishes.
Petri dish with comb made from
ice-cream container lid.
Comb removed, excess agar is cut
and electrodes added. Wells filled AFTER buffer is added.
FIND OUT MORE:-
In 2006 Michael created a DNA
Fingerprinting and DNA sequencing simulator that can also download and
run real DNA sequences from the NCBI Genbank database! It even reads
the sequence out loud...cool!
Identify which child is adopted in a
family of five, or which disease organism is in a patient sample or
identify which suspect was at the crime scene. Each time the
application is run a unique set of prints is generated. GeneE an
electrophoresis virtual lab & a DNA fingerprinting vritual lab.