Agarose Gel Electrophoresis of DNA
Exp. 4: Analysis of DNA Fragments Using Agarose Gel
Electrosphoresis
【Objective】
1. To learn and understand the characteristics of agarose gel electrophoresis and
its application to DNA analysis.
2. To become familiar with the experimental procedures of agarose gel
electrophoresis for DNA analysis
3. To become able to analyze DNA fragments using agarose gel electrophoresis.
【Principle】
DNA molecules have a high density of negative charge due to the phosphate
groups on the DNA backbone. Under an electric field, these negatively charged
DNA molecules migrate toward the positive end of the electric field. It is the case
of an agarose gel immersed in a buffer solution of pH 7.5.
In agarose gel electrophoresis, DNA molecules are forced to move through a
sieve of molecules made of agarose gel. As a result, DNA molecules are
separated based on their sizes. The larger the DNA fragments, the slower they
move.
Ethidium bromide (EtBr) is an intercalating dye, meaning that it inserts into
the space between the adjacent bases stacked in the center of the DNA helix,
forming EtBr-DNA complexes. Under the UV light (300~360nm) radiation, the
EtBr will emit fluorescence of orange color at 590nm. In the current experiment,
when the standard marker along with DNA samples is run on the same agarose gel
and stained with EtBr, 6 bands of standard DNA size at 23.1, 9.6, 4.4, 4.3, 2.2, and
2.0 kb will be observed, respectively. In comparison with these markers, the sizes
of DNA fragments could be determined accordingly.
【Experimental Materials】
1. 1% agarose gel
2. Electrophoresis buffer (1xTAE)
3. Standard DNA marker
4. EtBr
【Procedure】
1. Set up the electrophoresis equipment.
2. Prepare the electrophoresis tank. Seal the open ends of tank with tape to form
a mold.
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Agarose Gel Electrophoresis of DNA
3.
4.
5.
6.
Pour gel into the electrophoresis tank.
(1) Add 0.8g of agarose to 80 ml of 1xTAE buffer in a conical flask. Mark the
level of the solution and add extra 5.0 ml of dH2O. 【Note: Aqueous
solution will be evaporated in the next step.
To keep the
concentration unchanged, some H2O needs to add in the solution.
Experimental experience indicates that 5.0 ml of H2O is just
adequate.】
(2) Warm the agarose solution in microwave for 2 min using the moderate
power. 【Note: Check the solution level against the marked line on the
flask.】
(3) Let the solution Cool down to 60℃.
(4) Add 200 μl of EtBr (a final concerration is 1.0 μg/ml) to the solution.
Then pour it into a the sealed gel casting platform and insert the gel comb
on the top of the gel cast. 【Note: Wear gloves all the time to avoid
contacting EtBr directly.】
(5) When the agarose solution becomes gel form, remove the seal and take out
the comb.
Place the gel into the electrophoresis buffer in the
electrophoresis tank containing sufficient. Make sure that the solution
level is 1mm~3mm above the toe of the gel.
Load the sample into the wells with a pipettor. 【Note: The DNA samples
should be added near the cathode.】
Electrophorese
Electrophorese at 100V for 1~1.5 hours until the bromophenol blue dye from
the loading buffer has migrated to nearly 2/3 of the gel. 【Note: To connect
the electric wire to the correct outlets of the power supply.】
Observe
Lay the gel on the top of the UV light box to observe the bands under the UV
light (300~360nm). 【Note: UV light is harmful to eyes and skin. Do not
observe and expose for a long period.】
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