CHEM 161L Biochemistry Laboratory 2

1
st
Quarter SY 2010-2011

Experiment 03 Group 1 September 13, 2010 1 of 3

Agarose Gel Electrophoresis of DNA

1
Professor, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology;
2
Student, CHM161L / A11, School of Chemical Engineering,
Chemistry and Biotechnology, Mapua Institute of Technology

ABSTRACT
Agarose gel electrophoresis is used on both biochemistry and molecular biology as a tool to analyse DNA and RNA
qualitatively and as a separation technique of both. The gel acts as a molecular sieve where DNA and RNA move. Electric
current was applied to the gel and with the running buffer, as it provides ions that carry current all over the gel. Since DNA is
negatively charge molecule due to its phosphate backbone, it experiences repulsion with negative electrode placed on top of
the wells where DNA is placed and travels toward the anode (positive charge). Larger molecule has slower mobility than
smaller molecule thus, bands at the end has smaller molecular weight and vice versa. Several factors affects the mobility of the
DNA and these are: voltage applied, DNA conformation, concentration of agarose (larger or smaller pore of gel), buffer used
(ionic strength). The gel was stained for the bands to be visible and shall be viewed under U.V light. In the experiment, the DNA
sample should be run in an agarose gel but unfortunately theres no DNA sample present thus, the experiment was not
performed. The goal of the experiment is to familiarize the students to the principle of separation of electrophoresis and the
factors affecting it.
Keywords: Gel electrophoresis, agarose, DNA, mobility,

INTRODUCTION
Agarose gel electrophoresis is another type of
electrophoresis technique that is carry out in the field of
biochemistry and molecular biology. This is a method which
separates DNA and RNA according to their weight and
structure using an electric field applied to a gel matrix. The
gel matrix serves as a filter that when current is applied the
DNA moves toward the other end due to repulsion.
DNA fragments are placed into the gel matrix through its
wells and electric field is applied across the gel. The
orientation of the DNA slab is that the wells where the DNA
samples are loaded are near the cathode. Since DNA
molecules are negatively charged due to its phosphate
backbone, it will move through the gel toward the positive
end of the electric field due to repulsion with cathode as
current is applied. As different sized DNA fragments move
through the pores of the gel, longer DNA fragments will
have higher retention time compared to the DNA fragments
that is shorter. The bands that are farthest from the wells
have the shortest DNA fragments while, bands that are
closest to the wells have the longest fragments. Longer
fragments mean higher molecular weight. Thus, the mobility
of the DNA is directly proportional to it log molecular weight.
Having a DNA marker, can generate a standard for
identifying the molecular weight of an unknown DNA
sample. Thus, agarose gel electrophoresis also serves as
an analytical method.
There are several factors affecting the migration of the DNA
through the agarose gel. As mention, the size of the DNA
affects the migration rate, larger fragments has slow
mobility compared to smaller fragments. DNA conformation
also affects the migration. Circular plasmids will migrate
more rapidly compared with linearized DNA. This is
because plasmid is more compact and has smaller are
compared to linearized DNA. Another factor is the
concentration of agarose. Higher concentration of agarose
facilitate separation of smaller DNAs while, low agarose
concentration allow resolution of smaller DNAs. Voltage
applied to the gel can influence the migration of DNA. When
the voltage applied is increased, larger gragments migrate
proportionally faster than small fragments. Thats why most
agarose gel electrophoresis is carried out with 5 volts per
cm of the gel. Lastly, the buffer used can also affect the
migration. DNA fragments will have a different mobility at
different buffers depending to the buffers ionic strength.
Buffer provides inos that carry a current throughout the gel.
In the experiment, agarose gel electrophoresis was used to
separateThe aim of the experiment is to familiarize student
to the principle of agarose gel electrophoresis and to the
factors that affects its migration.
MATERIALS AND METHODS
CHEM 161L Biochemistry Laboratory 2
1
st
Quarter SY 2010-2011

Experiment 03 Group 1 September 13, 2010 2 of 3
Buffer Preparation
50 X TAE 10 X Tris-
phosphate
10 X TBE
Tris base 242
g
Tris base 108
g
Tris base 108
g
Glacial
HOAc
57.
1
85%
H3PO4
15.
5
ml
H3BO3 55
g
0.5M
EDTA(pH
8)
100
ml
0.5M
EDTA(pH
8)
40
ml
Na2EDTA.2H2
O)
9.3
g
Dilute to 1L Dilute to 1L Dilute to 1L

TAE working
solution
Tris-phosphate
working solution
TBE working
solution
Tris-
acetat
e
0.04M Tris-
phosphat
e
0.08M Tris-
borate(p
H 8.3)
0.089
M
EDTA 0.001
M
EDTA 0.002
M
Disodiu
m EDTA
0.025
M

50X TAE
242 g Tris base, 52.1 mL glacial acetic acid and 100 mL 0.5
M EDTA at pH 8 was mixed and the mixture was diluted to
1.0L distilled water
10X Tris-Phosphate
108g Tris base, 15.5mL 85% phosphoric acid and 40 mL
0.5 M EDTA at pH 8 was mixed and diluted to 1.0L distilled
water.
10X TBE
108 g Tris base, 55 g boric acid and 9.3 g NO2 EDTA .
2H2O was placed in a 1-L volumetric flask and diluted with
distilled water.
TAE-Working Solution
Gel Preparation (0.8% Agarose Gel)
0.4 g of gel powder was weighed and dissolved in 50 mL of
chosen buffer. The mixture was placed in microwave oven
and heated until boiling for one minute. After, the solution
was swirl and reheated for w minutes. The gel was allowed
to cool. 5 L of 5 mg/mL ethidium bromide was added to
the solution. The solution was poured carefully into the
casting tray so no bubble will form. The comb was placed
over the gel. The gel was allowed to polymerize. After, the
comb was removed in a fluid motion so the wells will be
preserved. Approximately 20 to 30 minutes, the gel
solidified. The wells were flushed with buffer before
samples were loaded.
Sample Preparation and Loading
Small piece of parafilm was cut and placed on table top. 1-3
L of loading buffer was pipetted out onto the parafilm. 5-
15 L of sample was added to the loading buffer on the
parafilm. The spot was drawn up and down using pipette for
the solution to mix well. The mixture was then loaded onto
the well using pipette carefully not to puncture it.
Running the Gel
30 L of 5mg/mL ethidium bromide was added to 300 mL
running buffer. The gel chamber was filled with running
buffer. The power supply or volts was set to 100V and
electrophoresis started. When the dye is 1 cm from the
edge of the gel slab, the apparatus was turned off.
Viewing the Gel
The gel was removed out of the chamber carefully not to
destroy it. Safety goggles were worn and the gel was
viewed under U.V light.
RESULTS
The experiment was not able to execute to due lack of
materials.
DISCUSSION
There is no separation or electrophoresis was performed.
This is due to lack of material --- DNA sample (fragments).
REFERENCES
CHEM 161L Biochemistry Laboratory 2
1
st
Quarter SY 2010-2011

Experiment 03 Group 1 September 13, 2010 3 of 3
1. Weaver, Robert F. (2001) Molecular Biology, 2
nd

Edition
2. Cox, Michael M., Nelson, David L. (2005)
Lehninger Principles of Biochemistry, 4
th
Edtion.
1. Garrett, Reginald H., Grisham, Charles M. (1999)
Biochemistry, 2
nd
Edition
2. Cox, Michael M., Nelson, David L. (2005)
Lehninger Principles of Biochemistry, 4
th
Edtion.
1. Weaver, Robert F. (2001) Molecular Biology, 2
nd

Edition