Agarose Gel Electrophoresis
Content
Introduction .............................................................................................................................................2
Materials for agarose gel electrophoresis .................................................................................................3
Method for agarose gel electrophoresis ..................................................................................................4
Preparing the gel .......................................................................................................................................4
Running the gel .......................................................................................................................................6
Visualize the gel ......................................................................................................................................9
1
Agarose Gel Electrophoresis
INTRODUCTION
In general, electrophoresis is a technique
that separates molecules on the basis of their
different rates of movement in an applied electric field through a porous semisolid matrix. The
semisolid matrix is a gel made from agarose and buffered water. Agarose gel electrophoresis is
commonest way of separating and analyzing DNA or RNA. This gel can be used to separate
and visualize DNA or RNA of various sizes.
PCR
This method also allows to determine whether
was successful, whether the resulting product is the correct size, whether other products
were amplified as well, and whether the concentration of the resulting product is suitable for cycle
sequencing. It has been observed that DNA fragments ranging from 50bp to 20,000bp are well
resolved using various concentrations of agarose gels. These characteristic features make this
technique to take the centre stage in molecular cloning experiments. Agarose is a polysaccharide
consisting of a linear polymer (repeating units) of D-galactose and 3,6-anhydro L-galactose.
Commercially, agarose is extracted from seaweed and purified for use in electrophoresis. The
movement of molecules through an agarose gel is dependent on the size and charge of molecules
and the pore sizes present in the agarose gel. Rates that molecules move through the gel can also
be effected by the sizes of pores in the agarose gel. Decreasing pore sizes increases the
separation of small and large molecules during electrophoresis.
2
Agarose Gel Electrophoresis
MATERIALS FOR AGAROSE GEL ELECTROPHORESIS
a. Agarose
a. Extracted from seaweed is a mixture of two components: the linear polysaccharide agarose,
and a heterogeneous mixture of smaller molecules called agaropectin
b. 10X TBE (Tris-Borate electrophoresis buffer)
OR
a. 108 g of Tris base [tris(hydroxymethyl)aminomethane] + 55 g of boric acid +7.5 g
of EDTA, disodium salt
c. 10X TAE (Tris-Acetate-EDTA)
a. 48.4g Trizma base + 20ml of 0.5M EDTA pH 8.0 + 11.44 ml glacial acetic acid
d. Gel loading dye
a. 50 mM EDTA + 0.2% SDS + 50% glycerol + 0.05% w/v bromophenol blue
e. Ethidium bromide (EtBr) (5 mg/ml)
a. Stain DNA by intercalating between the bases of DNA
f. Electrophoresis equipment
3
Agarose Gel Electrophoresis
METHOD FOR AGAROSE GEL ELECTROPHORESIS
1. Prepare 1% gel, for example; add 0.3 g agarose to 30 ml 1x TBE
2. Heat the solution to boiling in the microwave to dissolve the agarose.
a. You should not see any beads in the solution. If you have beads in the agarose,
bands are get distorted.
4
Agarose Gel Electrophoresis
3. Add 5 μl of ethidium bromide to the dissolved agarose and mix.
a. Ethidium bromide is an intercalating agent commonly used as a fluorescent tag. It is commonly
used to detect nucleic acids in molecular biology laboratories.
b. a powerful mutagen so ensure that it does not get spread around the lab.
4. Prepare the electrophoresis system.
a. Place gel tray into casting chamber.
b. Add casting comb(s) into the appropriate slot(s)
5
Agarose Gel Electrophoresis
5. Pour agarose into gel tray to about 5-7mm.
6. Wait sitting for at least 30 min, until gel is cool to touch and is opaque in appearance.
7. Once set, place gel and tray into gel rig, with wells on the left (cathode) side.
8. Fill gel rig with 0.5X TBE sufficient to cover the entire gel.
9. Carefully remove combs by pulling them upwards firmly and smoothly in a
continuous motion.
6
Agarose Gel Electrophoresis
10. Cut a piece of parafilm and place it flat on the bench top.
11. Using a pipette, place small dots of 6X loading dye (about 1-2uL) onto parafilm, in
rows of 8, 1 dot for each PCR sample that will loaded on the gel.
12. Using a pipette take 3 μl of product and pipette it onto its corresponding dye dot, then
mix the sample and dye by pipetting up and down. Then, pipette up the dye/sample
solution and pipette it into the proper well
13. Put gel box cover into place (this step is essential for your gel to run and to minimize
the risk of electric shock).
14. Turn on power supply. Run the gel for 75 minutes at 80 volts.
7
Agarose Gel Electrophoresis
15. Check for bubbles at the cathode side to ensure that gel rig is running.
16. Tips to improve your gels
a. Do not shake the gel chamber while loading as this will facilitate diffusion of
samples out of the wells
b. Insert pipette tips into wells at an angle to avoid puncturing the well, which
will result in loss of sample.
c. Always make sure that your gel is running before leaving.
d. Work as fast as you can without sacrificing accuracy and safety. The longer
your samples sit in the buffer, the more diffuse your bands will bewhen you
visualize the gel.
e. Until you are comfortable, only load 1 row of samples at a time, run the gel for
5 minutes, then load the next row of samples. This will minimize sample
diffusion.
17. Turn off power supply. Using gloved hands, remove the cover from the gel box.
18. Remove the gel and casting tray from rig, avoiding dripping buffer all over the
benchtop.
8
Agarose Gel Electrophoresis
19. Put on UV protecting face shield and turn on UV light box to see stained sample
bands and DNA ladder. Ensure that all around you are protected for UV exposure.
9