Introduction:
The
size of a DNA fragment can be estimated by gel electrophoresis. This technique separates fragments by charge,
size (molecular weight) and shape.
First, an agarose gel is made with slots (wells) in it. The DNA sample is dispensed in the well, a
buffer solution is placed in the apparatus, and an electric current is run
through the gel. DNA molecules are
negatively charged due to the phosphates in its backbone, and when placed in an
electric field starting at the negative (black) electrode, it will migrate
towards the positive (red) electrode.
The gel is a complex molecular network containing narrow passages. Smaller DNA molecules pass through more
easily (less friction) and migrate faster through the gel than larger size
fragments. Linear molecules also
migrate faster through a gel, compared to globular forms.
Since
the DNA fragments generated in this experiment are all negatively charged and
linear in shape, the only variable to observe during electrophoresis is
size. To determine the size of a
molecule, a standard or positive control is run concurrently in the gel. The standard consists of fragments of DNA of
known size. A positive control contains
the same size fragment (in our case 662 bp) as potential positive samples.
Gel
Electrophoresis
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DNA
fragments are viewed under ultra violet light, and a brightly colored band
indicates the presence of DNA. Ethidium
bromide (EtBr) is used in the gel to view the DNA. The gel is stained with EtBr
after the electrophoresis run, or is placed in the gel prior to gel
solidification.
EtBr is an intercalating agent that fits into the middle of
the DNA molecule, causing it to bulge.
UV light fluoresces the bulging areas of the DNA, which gives it a
bright appearance.
EtBr is a mutagen and possible
carcinogen. Therefore, it is extremely
important that you only handle the compound with gloves. If EtBr is accidentally spilled, immediately
ask for assistance to clean it up.
Materials:
micropipettors vortexer microwave
pipette tips gloves
(3 sizes) rubber
hothands
waste beaker microfuge weigh balance
microtube rack agarose
loading dye chloroform TAE
buffer
125 ml bottle / funnel power supply electrophoresis equip.
Procedure:
I) Prepare a 1% agarose
gel
II)
Prepare the gel for loading
III)
Prepare the samples for loading
IV) Perform
gel electrophoresis
V)
View PCR products under ultraviolet
light
I.
Prepare 1% Agarose Gel:
(with gloves)
1)
Place the funnel in a 125 ml
bottle and place on the balance. Press tare.
2)
Pour 0.5 g of agarose down
the funnel.
3) Add
50 ml 1X TAE buffer to the funnel. Try
to get all residual agarose from the sides of the funnel into the bottle. [TAE = Tris acetate EDTA]
4) Close
cap and shake to make sure no agarose is stuck to bottom of the bottle.
5) Loosen
cap and microwave for 1 min
only. If solution starts to boil, stop
the microwave immediately to avoid spillage.
Clean microwave tray if this occurs.
6)
Swirl gently using a hothand
and microwave for 10 seconds more.
7) While
waiting for the gel to cool, place the gel mold (that has side gaskets) in the
middle of the electrophoresis chamber so that the gaskets are tightly fitted to
the sides to avoid leakage.
8)
If the agarose bottle can be held
without discomfort, then continue. (Do not wait too long. Otherwise, the gel will start to
solidify). With gloves, get ethidium
bromide from instructor and, using a sterile white tip, add 1 ml EtBr
(be careful).
9)
Swirl until you do not see
the red EtBr.
10) Pour
into mold and place 2 rows of combs, on top and in the middle.
11)
Rinse out bottle immediately
before the excess agarose solidifies.
II. Prepare
Gel for Loading: (with gloves)
1) After
gel has solidified (takes about 15-20 min), pull gel mold up and out of
chamber.
2) Place
gel into electrophoresis apparatus, with the top row at the end with the black
(negative) electrode.
3) Add
1X TAE to both sides of the chamber until there is a small amount above the gel
(touch top of gel with finger to judge). Do not over pour.
4) Take
out combs carefully, by lifting straight up.
The buffer on top of the gel helps the combs to slip out without tearing
the gel.
III. Prepare
Samples to load in Gel: (with gloves)
1)
Spin samples briefly and
place tubes in rack. Open tubes.
2)
Using the same white pipette
tip, add 4 ml loading dye
to each cap.
3) Using
the same blue pipette tip, add 250 ml chloroform
to each tube. Close each cap as soon as
you add the chloroform (it vaporizes quickly).
4) Pick
up 2 tubes at a time, shake quickly up and down, and place in microfuge; Do the same for all of the tubes.
5) Spin
briefly. The chloroform dissolves the
oil. The PCR mix is now the blue bubble
floating at the top.
6) Load
~25 ml of each
sample into each well. Each group has
its own row to load. Make sure you draw
a picture indicating which wells contain which samples.
IV. Gel
Electrophoresis:
1) Slide
the lid on top. Make sure you do not
spill any buffer. If you do, then use a
kimwipe to dry it up. You do not want to
get electrocuted when it is turned on!
2) Place
the black electrode into the black hole of the power supply. Place the red electrode into the red
hole. Turn on power supply. Set the electric current to 80-100
volts. Press the runner button to start.
3) In
15 minutes, check to see how far the dye has migrated in the gel. If it is midway between wells (or from the
end for the second set of wells), then turn the power off.
V. Viewing
of PCR Products under U.V. Light:
(with gloves)
1)
Carefully slide the lid
off. Try to avoid spilling the buffer.
2) Grab
both sides of the gel that have the gaskets and lift. Holding these sides will avoid the gel from
sliding off. Tilt to pour off any extra
buffer.
3) Place your gel inside the
photodocumentation system using the U.V. box.
4) If
possible, capture an image of the gel using the photodocumentation system. Save your image on a zip disk.
5) Print out your gel image using the
photodoc system.
6) Lift the Plexiglas lid of the
transilluminator and place the gel on top.
Close the lid over your gel and put on goggles. Cut out the PCR bands that were positive
(except for the positive control). Use a
razor blade and gently cut down on all four sides of each band. Using the razor blade, flip the band on its
side and trim off the bottom, where there is no sign of DNA. Place the band in a 1.5 ml tube. Label the tube with your initials and the
sample number. You will now prepare each
sample for cycle sequencing.