Making an Agarose Gel Electrophoresis Equipment

Flask for boiling

Agarose
 Gel casting tray and combs

Gel combs specifies the length and width of the electrophoresis well to be used.
Place a casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.
 Step 1 Step 2

Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.
Buffer for agarose electrophoresis

• Tris/acetate buffer (TAE)

- Suitable for short runs

- Good large DNA fragments

- Better for downstream application

• Tris/borate (TBE)

- Suitable for long runs

- Good for < 2kb fragments

- May affect downstream apps (enzymatic, purification)

 Melting the Agarose

Agarose is insoluble at room temperature (Left)

The agarose solution is boiled until clear (Right)
Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve.
Be careful when boiling – the agarose solution may become superheated and may boil violently if it
has been heated too long in a microwave oven.
Agarose concentration

Agarose (%) Effective range of resolution of linear DNA fragment (kb)

0.5 30 to 1

0.7 12 to 0.8

1.0 10 to 0.5

1.2 7 to 0.4

1.2 3 to 0.2

Current Protocols in Molecular Biology 2003

 Pouring the gel

 
Allow the agarose solution to cool slightly () and then
carefully pour the melted agarose solution into the
casting tray. Avoid air bubbles.
When cooled, the agarose polymerizes, forming
a flexible gel. It should appear in color when
completely cooled (30 – 45 minutes). Carefully Add enough electrophoresis buffer to cover the gel to a depth of at
least 1 mm. Make sure each well is filled with buffer.
remove the combs and tape.
 Sample Preparation

Mix the samples of DNA with the 6X sample

loading buffer (w/tracking dye). This allows the
Carefully place the pipette tip over a well and gently expel
samples to be seen when loading into the gel and
the sample. The sample should sink into the well. Be
increases the density of the samples, causing them
careful not to puncture the gel with the pipette tip
to sink into the gel wells.
6X loading buffer:
- Bromophenol Blue (for color)
- Glycerol (increase the density of the mixture)
 Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the
electrical leads to the power supply. Be sure the leads are attached correctly – DNA migrates
toward the anode (red). When the power is turned on, bubbles should form on the electrodes in
the electrophoresis chamber.
 Cathode (-)

DNA (-) Well

Bromophenol Blue

 Voltage: 4-10 V/cm

Anode (+) Large fragment: V/cm, 0.5% agarose

After the current is applied, make sure the gel is running in the correct direction. Bromophenol blue
will run in the same direction as the DNA.
 DNA Ladder Standard

Dye 1% agarose, bp 2% agarose, bp

Xylene Cyanol 4000 1000

Cresol Red 1500 300

Bromophenol Blue 400 150

Orange G 50

Amaranth < 50 < 10

Relative electrophoretic mobility of leader dye in agarose

Bromophenol Blue

Inclusion of a DNA ladder (DNAs of know size) on the gel makes it easy to determine the sizes of unknown DNAs.
Staining the Gel
- After electrophoresis is complete, we can not see the DNA because the DNA is not visible to the naked eyes.
- Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel.
- Ethidium bromide can be added to the gel and running buffer before the gel is run (or the gel can be stained after it
has run).

Notice: Ethidium Bromide is a powerful

mutagen and is moderately toxic. Gloves
should be worn at all time.
 Ethidium Bromide requires an
ultraviolet light source to visualize

Place the gel in the staining tray containing warm

diluted stain.
Allow the gel to stain for 25 – 30 minutes.
To remove excess stain, allow the gel to destain in
water.
Replace water several times fro efficient destain.
When turned on, the UV rays will shine through the gel and will
stimulate Ethidium Bromide molecules in the luminescent gel.
Safer alternatives to Ethidium Bromide
The dye does not penetrate the cell membrane so it is not toxic to the
cell’s pharmacological activities.
If PCR products are used after electrophoresis to induce inflow into
bacteria, new generation dyes should be used because they are not
toxic to bacterial cells.
• Advantages
- Safer
- Ultra – sensitive
- Extremely stable
- Simple to use
- Compatible with standard instruments
- Compatible with downstream apps (cloning, expression)
• Disavantages
- More expensive