MIRAMAR COLLEGE
Life Sciences Department
Introduction to Biological Science (Biol210A)
Instructor: Elmar Schmid, Ph.D.
Agarose Gel Electrophoresis
Objectives
• To introduce you to agarose gel electrophoresis a method commonly used for
separation of DNA fragments
• To learn size determination of DNA fragments (standard curve) after gel
electrophoresis
• To learn to pour agarose gels.
• To analyze and size DNA fragments by agarose gel electrophoresis
Introduction
The agarose gel electrophoresis method is widely used in the biotechnology industry for
the routine analysis of nucleic acids, most importantly DNA. This technique separates
molecules based upon charge, size and shape. It is particularly useful in separating
charged macromolecules such as DNA and RNA. Proteins can also be separated by
agarose gel electrophoresis although this is not a common practice. Proteins are
routinely separated by another gel electrophoresis method called SDS-PAGE.
Agarose gel electrophoresis possesses great resolving power, yet is relatively simple
and straightforward to perform. The gel is made by dissolving agarose powder (a red
algae-derived polysaccharide) in boiling buffer solution. This solution is then cooled to
approximately 50°C and poured into a mold (gel cast) where it solidifies like gelatin. A
special-shaped Teflon comb is used to make depressions in the gel for loading samples
for analysis. Prior to loading samples, the tray - holding the solidified agarose gel - is
then submerged in a buffer-filled electrophoresis chamber which contains electrodes- a
submarine gel apparatus.
DNA samples are prepared for electrophoresis by mixing them with solutions of sample
buffer containing glycerol or sucrose. This makes the samples denser than the
electrophoresis buffer. These samples can then be loaded with a micropipette into
wells that were created during gel forming process by a comb. The dense samples sink
through the buffer and remain in the wells. Sample buffer also often contains
bromophenol blue to help see the sample and to track sample migration through the gel.
A direct current (DC) power supply is then connected to the submarine gel apparatus
and a constant electrical current is applied. Charged molecules in the sample enter the
gel through the walls of the wells. Molecules having a net negative charge migrate
towards the positive electrode (anode) while net positively charged molecules migrate
towards the negative electrode (cathode). Within a range, the higher the applied
voltage, the faster the samples migrate. The buffer in the submarine gel apparatus
conducts electricity and controls pH. The pH is important to the charge and stability of
biological molecules.
Agarose is a polysaccharide derivative of agar. The agarose gel contains microscopic
pores which act as a molecular sieve. The sieving properties of the gel influences the
rate at which a molecule migrates. Smaller molecules move through the pores more
easily than larger ones. Molecules can have the same molecular weight and charge but
different shapes. Molecules having a more compact shape (ex. a sphere is more
1
MIRAMAR COLLEGE
Life Sciences Department
Introduction to Biological Science (Biol210A)
Instructor: Elmar Schmid, Ph.D.
compact than a rod) can move more easily through the pores than molecules which
cover more molecular space.
Analysis of DNA often involves determination of the length of fragments of DNA. Length
is measured as number of base pairs for small molecules and kilobase pairs for large
ones. One kilobase pair equals 1000 base pairs. By comparing the length of unknown
DNA to pieces of known size (standards or DNA markers) the number of base pairs can
be measured. Conveniently, this can be determined with agarose gel electrophoresis.
A ladder is standardly used and a standard curve of distance migrated versus kilobase
length can be constructed. Using the standard curve, the kilobase length of an
unknown can be determined from the distance migrated.
Also, certain dyes can be used as standards to calibrate an agarose gel. Dyes such as
xylene cyanol, bromophenol blue and orange-G are fairly accurate size markers for
small DNA fragments. On a 1.2% agarose gel these dyes migrate as follows:
Dye
Xylene Cyanol
Bromophenol blue
Orange-G
Color
Base Pair Equivalent
blue-green
2800
purple-orange
250
orange
70
In this exercise, the principles of agarose gel electrophoresis will be learned. You will
also learn how to analyze the length of DNA (in base pairs) using a standard curve
derived from an agarose gel.
origin
Use this picture of a gel to determine
how to practice drawing a standard curve.
Standard
Distance from
origin
5 kb
3 kb
2 kb
Plasmid
DNA
2
MIRAMAR COLLEGE
Life Sciences Department
Introduction to Biological Science (Biol210A)
Instructor: Elmar Schmid, Ph.D.
DNA STANDARD CURVE
10000
BASE PAIRS
1000
100
10
1
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
Dis tance M igr ated
Use the graph to determine the size of the plasmid DNA.
3
7.0
7.5
8.0
8.5
9.0
9.5
10.0
MIRAMAR COLLEGE
Life Sciences Department
Introduction to Biological Science (Biol210A)
Instructor: Elmar Schmid, Ph.D.
Procedure
1. Pour a 1% agarose gel using 1X TBE (Tris-borate-EDTA buffer).
-
-
-
-
-
Weight in 0.5 gm of agarose powder, using a weighing boat.
Transfer powder to a clean and dry 250 ml Erlenmeyer flask
Add 50 ml of 1X TBE buffer and gently swirl the suspension
Heat the 1% agarose mixture by using the microwave - set at high
temperature - for approximately 1 minute (maybe 1:15 min)
- you know the agarose has totally melted once it bubbles and no
suspended particles are seen.
Use thermogloves and take the flask (containing the melted agarose) out of
the microwave and place on your bench; gently swirl the content without
introducing pubbles!
Allow to cool for 5 – 10 minutes (glass should be hand-warm)
Put on plastic gloves and pipette 5 l of a 10 mg/ml Ethidium Bromide (EtBr)
solution to the melted agarose solution; gently swirl the flask
(Attention! Be extremely careful not to get in touch with the solution!
Ethidium Bromide is a known mutagen and is suspected to cause cancer!!!!)
Place a Teflon comb in the notches of the gel tray provided, raise the top
and bottom side of the tray, secure, and pour the melted agarose into the
gel tray.
Allow to set or solidify at room temperature.
Top with 1X TBE electrophoresis buffer ~ 250-300 ml, until the gel is totally
submerged.
2. Sample preparation
- Mix 5 l of sample A (containing two DNA fragments) with 1 l of loading dye.
3. Load the gel
Use a micropipet to load 2 lanes:
Lane 1: 5 l of the 1 Kb (kilo base) ladder to which loading dye has already been
added, as a molecular weight marker.
Lane 2: 6 l of sample A to which you have already added the
loading dye.
4. Load 2 wells of a gel that you will share with other students and
don’t forget to include a molecular weight marker in one of the
lanes.
5. Run the gel at 100 volts for approximately 30 minutes.
6. Visualize under UV trans-illumination. Use the proper goggles to
avoid the harmful effects of UV light.
4
MIRAMAR COLLEGE
Life Sciences Department
Introduction to Biological Science (Biol210A)
Instructor: Elmar Schmid, Ph.D.
7. Determine the size of each of the two DNA fragments present in
sample A.
Size of DNA fragment in
standard (base pairs)
Starting from the shortest*
Distance of standard
fragments from origin (cm)
Distance traveled by each
of the DNA fragments
present in the test sample
(cm)
2000
1650
1000
850
650
500
400
300
200
100
(*) The standard does have higher molecular weight fragments that can not be properly
resolved on a 1% gel under the running conditions.
5
MIRAMAR COLLEGE
Life Sciences Department
Introduction to Biological Science (Biol210A)
Instructor: Elmar Schmid, Ph.D.
DNA STANDARD CURVE
10000
BASE PAIRS
1000
100
10
1
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Dis tance M igr ated
Attach a picture of your results and explain them.
6
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0