Agarose Gel Electrophoresis
Molecular Biology Lab #4
Background:
Electrophoresis through agarose or polyacrylamide gels are important techniques that are
used to separate and purify nucleic acids. Agarose gels are used most commonly, and will
be emphasized in this lab. Acrylamide gels are useful for separating very small fragments
of DNA (5 to 500 bp). Their resolving power is very great and they allow one to separate
fragments that differ in size by only one base pair (i.e. DNA sequencing gels). However,
polyacrylamide gels are also more difficult to prepare and handle than agarose gels.
Although agarose gels have a lower resolving power, they have a much greater range of
separation.
Agarose is a linear polymer of D-galactose and L-galactose. The polymers form helical
fibers that aggregate into supercoiled structures. These form a three dimensional mesh of
channels with diameters ranging from 50 to 200 nm. The greater the pore size of the gel,
the larger the DNA molecules that can be fractionated. DNA is usually separated in
agarose gels that are constructed in a horizontal format. A constant electrical field is
applied and the negatively charged DNA migrates to the positively charged terminal.
Large DNA molecules migrate more slowly than small ones because it is more difficult
for big molecules to ‘worm’ their way through the pores.
There are several factors that determine the rate of DNA migration through the agarose
gel.
1. The molecular size of DNA: Molecules of double stranded DNA migrate through
agarose gels at rates that are inversely proportional to log 10 of the number of base
pairs. Large molecules move more slowly than small ones because of frictional
drag and the difficulty of moving through the smaller pores of the agarose.
2. The concentration of agarose: There is a linear relationship between the log of
electrophoretic mobility of DNA and the gel concentration. The more
concentrated the gel, the more slowly the DNA molecules migrate.
3. Conformation of DNA: Plasmid DNA has at least three conformations. These are
supercoiled (form I), nicked circular (form II), and linear (form III). Supercoiled
DNA is a circle that coils tightly on itself to form a dense structure. Nicked
circular DNA has one strand nicked, allowing the supercoil to be relaxed. Linear
DNA has 2 nicks. These forms all can exist in a single plasmid prep. The
migration pattern of the three forms is complex and depends on the concentration
of agarose and the strength of the electrical field. The best way to distinguish
form III DNA is to cut the plasmid DNA with a restriction nuclease and separate
the digested DNA in a lane adjacent to the native or undigested DNA. This will
identify the linear form.
4. The presence of ethidium bromide: Minigels are frequently run with ethidium
bromide incorporated into the agarose and/or electrophoresis buffer. Intercalation
of the dye causes a decrease in the negative charge of the DNA and inhibits the
rate of migration by about 15%.
5. The applied voltage: At low voltage, the rate of migration of DNA through the gel
is directly proportional to the voltage applied. However, as the voltage is
increased, the migration rate of large DNA changes differentially relative to small
fragments. High voltage also generates more heat, which can deform or melt the
gel.
6. Electroendo-osmosis (EEO): Ionized acidic groups are often attached to the
polysaccharide matrix of agarose gels. When voltage is applied, these ions move
in the opposite direction of DNA (to the negative pole). The more acidic groups in
the agarose, the greater this problem becomes. It is always best to buy good
quality agarose with low EEO flow.
Several different gel running buffers are used for gel electrophoresis: One of the most
common is TAE (Tris acetate and EDTA). The ionic strength of the buffer influences
migration in the gel. Very low ionic strength (oops, too much water) results in incomplete
migration, whereas, high ionic strength (Oops, forgot to dilute the 10x stock) results in
rapid migration but excess production of heat. This mistake can lead to melting of your
gel.
The other gel buffer is gel loading buffer (sometimes called blue juice). This is mixed
with the DNA sample just before loading wells. Gel loading buffer is important for three
reasons. The glycerol, sucrose, or ficoll makes it heavier than water so the sample sinks
evenly into the well. The bromophenol blue or cyanol add color, which facilitates sample
loading into wells and tracking the dye as it migrates through the gel. Bromophenol blue
migrates about as rapidly as a 300 bp DNA fragment and provides a warning about when
to stop electrophoresis (Oops, my DNA ran off the end of the gel).
After electrophoresis, the positions of the restriction fragments are determined by staining
with ethidium bromide. This is a dye that intercalates between the DNA strands and emits
fluorescence at 590 nm as red orange light. Ethidium bromide can be used to detect either
RNA or DNA, but it is much more sensitive for DNA (can detect as little as 10 ng). The
gel can be stained after electrophoresis, or ethidium bromide can be incorporated into the
gel while it is running.
Another molecule, SYBR gold, is even more sensitive than ethidium bromide. However,
SYBR gold is very expensive. Therefore, it is not routinely used for staining bands in
agarose gels.
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Objective:
The objective of this lab is to provide an introduction to agarose gel electrophoresis. Each
group will set up a mini gel and use this to fractionate the restriction digest from the
previous lab period.
Materials:
Minigel boxes
Power packs
Microwave oven, 0.8% agarose in TAE buffer.
Tape
DNA 1 kb ladder molecular weight marker
Plastic trays for washing gels.
TAE buffer
Micropipettes and yellow tips
6x gel loading buffer
ethidium bromide stock solution (10 mg/ml).
UV light box
Photographic equipment
Procedure:
1. Seal the edges of the gel form with tape and run your finger over the tape to make
sure that the seal is tight. Set the form on a level surface. Insert the comb in the
slots on either side of the form. Make sure that it is evenly aligned and that about
1 mm free space exists between the bottom of the form and the bottom of the
comb. The comb should be on the black side of the gel box (- side) so that
samples can migrate to the red (+ side).
2. Microwave the 0.8% agarose at full strength. Make sure that the cap of the
bottle is loose to allow pressure to be released (yes, it will explode if the cap is
tight). Keep a close watch during microwaving. If the agarose starts to boil
strongly, turn off the oven temporarily. After microwaving for 1-2 minutes, use a
glove to remove the bottle of agarose and gently mix. Be very careful not to
shake too hard and cause violent boiling over. When the agarose is liquid and
completely clear, place the bottle in a water bath at 65ºC to cool.
3. When the bottle is cool enough to touch comfortably, add ethidium bromide to a
final concentration of 0.5 g/ml (about 5 l / 100 ml). Be careful and use gloves
because ethidium bromide is a carcinogen. Mix contents.
4. Pour the gel when the bottle of agarose is cool enough to handle with your bare
hand. If it is too hot, you may cause the plastic gel form to warp. Pour the agarose
solution slowly into the form until the level is about 5mm thick. Check that no
air bubbles are lodged beneath the comb (jiggle it from side to side). Thicker
gels are sturdier and easier to work with, however, the bands are not as sharp as in
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thinner gels. Thin gels (3mm or less) are difficult to work with and they tear more
easily. Allow the gel to polymerize for at least 30 min before use.
5. Add just enough TAE buffer to cover the gel to a depth of 1 mm. Gently remove
the comb by picking up one end and slowly removing from the gel. Do not go too
fast. Removing the comb too quickly can tear the gel or cause the wells to
collapse.
6. Cover the minigel box and hook the black and red electrodes to the terminals on
the power packs. Turn on the voltage to 90 and carefully observe the electrode
wire in the bottom of the minigel box. There should be small air bubbles if the
power pack and all connections are OK. Turn off power and remove the top of
the gel box.
7. Heat the restriction digests in a 65ºC water bath for 4 min. Place samples in a
small rack and allow cooling for 5 min.
8. Add 3 l of 6x gel loading buffer to each tube. Set the micropipetter to 20 l and
mix the gel buffer and sample. Try to avoid introducing air bubbles.
9. Place a dark paper directly beneath the wells to allow better visualization.
Carefully add 10 l of sample (about 1 g of DNA) to each well. Be careful not to
allow the sample to overflow during filling. Add 3 l of the 1 kB ladder of DNA
molecular weight markers to the last well.
10. Close the lid of the gel box and turn the power pack on to 90 V. Do not stick
your fingers inside the gel box or you might get a big shock!! You should see
bubbles at the electrode due to electrolysis. After one or two min, the
bromophenol and xylene cylanol dyes should begin to migrate toward the positive
terminal. Turn down the voltage and allow the gel to run for 1 hour.
11. After 1 hour, the blue markers should have migrated about ¾ of the length of the
gel. At this point, turn off the power pack and remove the top cover of the
minigel. Carefully remove the gel form containing the minigel (use gloves as
ethidium bromide is a potent mutagen) and transfer it to a plastic dish containing
1x TAE buffer. Tip the form to allow the gel to slide into the buffer. Mark your
gel by cutting corners off.
12. Pick up the gel using a spatula and transfer it to a UV light source. Cover your
eyes with goggles, as UV is harmful. Turn on the UV light to visualize the DNA
restriction pattern. You should see a ladder of bands in the lane loaded with
molecular weight markers.
13. Photograph the gel using the Kodak digital camera and save a picture to a floppy
disc to print for your lab notebook. Sometimes the files will not open. Make sure
that you check your file to confirm that it will open.
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14. Dispose of the stained minigel in the orange biohazard bag. Pour the buffer from
each gel box into the waste bottle using a funnel. Wash your minigel box in tap
water and invert to dry on a paper towel.
15. Clean up your lab bench, and put away materials!
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