Gel Electrophoresis

What is electrophoresis?
electro = flow of electricity
phoresis (Greek) = to carry across

 A molecule with a net charge will migrate in an

electric field
 Gel electrophoresis is using an electric current
to cause a charged particle to move through a
non-reactive matrix or molecular sieve (gel)

 A gel is a colloid, a suspension of tiny particles

in a medium, occurring in a solid form, like
gelatin
 How does it work?
DNA is an organic acid, and is negatively charged
(remember, DNA for Negative)

When the DNA is exposed to an electrical field, the

particles migrate toward the positive electrode

Smaller pieces of DNA can travel further in a given time

than larger pieces

The gel is then visualized

 Gel Visualization
• Gel is visualized under UV
light

• UV light causes stained DNA

to fluoresce

• The most common

fluorescent dye for staining
DNA is Ethidium bromide (EB)

• Stained DNA appear as bands

under UV light
Under UV light
Clear and sharp bands

bad gel
 Factors affecting DNA migration

• The velocity of migration of a molecule in an electric

field depends on:
–the electric field strength (voltage applied)

–electrophoretic mobility of the molecule

»size of the molecule
»concentration of gel
»pH and ionic strength of electrophoresis
buffer
 Materials required for electrophoresis

• Agarose or polyacrylamide
• Buffer ( TAE or TBE): The buffer provides
ions in solution to ensure electrical
conductivity.
Acetic acid/Boric acid

• Power supply and a gel chamber

Protocol for Agarose Gel Electrophoresis

Agarose is a polysaccharide purified from seaweed.

Materials required for agarose gel

Agarose
0.5×TBE buffer*
Tracking dye
Loading aids
Electrophoresis chamber
Power supply
Gel casting tray and combs
EB (DNA staining)
Gloves
Pipette and tips
*TBE buffer: Tris-base, Boric acid, EDTA
 Procedure: 1% agarose Gel
 Prepare a casting tray

 Suspend dry agarose in a buffer solution (TAE or TBE): 1g for 100ml Buffer

 Boil until the solution becomes clear, add EB and mix well

 Pour it into the casting tray, put comb(s) and allow it to cool

 Submerse the gel in a chamber containing a buffer solution, and remove comb(s)

 Load DNA samples

 Run for ~30min and take pictures

 Protocol for Gel Electrophoresis: 6% polyacrylamide
Materials required for polyacrylamide gels for separating DNA
fragments
29:1 Acrylamide/bis-acrylamide
10% APS: ammonium persulfate
TEMED: an oxygen scavenger (-N,N,N',N'-tetramethylethylene diamine
0.5×TBE buffer, Electrophoresis chamber, Power supply, Gel plate sets
and combs, EB, Gloves, Pipette and tips
 Protocol for Gel Electrophoresis: 6% polyacrylamide

“Polyacrylamide is formed by the polymerization of

the monomer molecule-acrylamide cross-linked by
N,N'-methylene-bis-acrylamide (BIS). Free radicals
generated by APS and a catalyst TEMED are
required to start the polymerization since
acrylamide and BIS are nonreactive by themselves
or when mixed together.”
 Protocol for Gel Electrophoresis: 6% polyacrylamide

I. Gel Preparation

• Clean and completely dry the glass platescombs, and any other
pertinent materialswith alcohol; and arrange gel mold

• Prepare acrylamide gel mixture

• Carefully pour the gel solution into the mold

• Place comb into gel mold and gel solution

• Wait for acrylamide solution to set up and become firm in the gel
mold (1.5 hrs or longer)
Polyacrylamide gel apparatus
 Protocol for Gel Electrophoresis: 6% polyacrylamide
II. Gel Electrophoresis

• Place gel into electrophoresis machine and remove bubbles

• Pre-run the gels for 60-100min at 300 volts

• Load PCR products with a pipette

• Connect the black cord (-) to the end closest near the well. The red cord
(+) should be the furthest from the wells holding the DNA samples

• Turn on the power supply that's connected to the electrophoresis

• About 2.5 hrs later, disconnect all power supply

• Remove the gel and place it on a UV light box

• Take picture(s)