Horizontal Electrophoresis
ProtocolBulletin 6206

Agarose Gel Preparation for DNA Separation
Agarose gels can be used to separate DNA of various sizes.
Before casting an agarose gel determine the appropriate
percent agarose gel to use, based on the size of DNA tobe separated.
Procedure
1.	Determine the amount of agarose (grams) required tomake the desired agarose gel concentration and volume.
Use tables below as a guide for agarose concentrationand gel volume requirements.
Example: For a 1% agarose gel, add 1 gram of agarose to
100 ml of 1× electrophoresis buffer.

Gel volume requirements.
Gel Size (thickness)

0.25 cm

0.5 cm0.75 cm

1.0 cmBase				7 × 7 cm

10 ml

20 ml

30 ml

40 ml

15 × 7 cm

20 ml

40 ml

60 ml

80 ml

15 × 15 cm

50 ml

100 ml

150 ml200 ml25 × 10 cm 		

125 ml185 ml

250 ml25 × 15 cm		185 ml

280 ml375 mlTray				7 × 7 cm

10 ml

20 ml

30 ml

40 ml

7 × 10 cm

15 ml

30 ml

45 ml

60 ml

15 × 7 cm

20 ml

40 ml

60 ml

80 ml

15 × 10 cm

30 ml

60 ml

90 ml

120 mlGel concentration required for DNA separation.

15 × 15 cm

50 ml

100 ml150 ml

200 mlGel Concentration (%)

DNA size

15 × 20 cm

70 ml

140 ml210 ml

280 ml0.50

1–30 Kb

15 × 25 cm

90 ml

180 ml

270 ml360 ml0.75

800–10 Kb

25 × 10 cm		125 ml185 ml

250 ml1.00

500 bp–10 Kb

25 × 15 cm		185 ml

280 ml375 ml1.25

400 bp–7 Kb

25 × 20 cm		250 ml375 ml500 ml25 × 25 cm		

310 ml465 ml625 ml*1.50

200 bp–3 Kb

2.00*

100 bp–2.5 bp

3.00*

40 bp–2Kb

4.00**

10–400 bp

Sieving agarose such as Certified™ PCR agarose** Sieving agarose such as Certified low range ultra agarose

2.	Add the agarose to a suitable container (e.g., 250 ml
Erlenmeyer flask, Wheaton bottle, etc.). Add theappropriate amount of 1× electrophoresis buffer andswirl to suspend the agarose powder in the buffer.
3.	The agarose can be melted by boiling on a magnetic
hot plate or in a microwave oven.

4a.	Magnetic hot plate method — add a stir bar to theundissolved agarose solution. Heat the solution to boilingwhile stirring on a magnetic hot plate. Bubbles or foamshould disrupt before rising to the neck of the flask.
4b.	Microwave oven method — place the gel solution into
the microwave. Using a low to medium setting, set thetimer for a minimum of 5 min, stopping the microwave
oven every 30 sec and swirling the flask gently to suspend
the undissolved agarose. This technique is the fastest andsafest way to dissolve agarose. Boil and swirl the solution
until all of the small translucent agarose particles aredissolved. With the small flask still in place, set aside tocool to 60°C before pouring.
Casting Agarose Gel Slabs
There are several ways to cast agarose submarine gels using
the Sub-Cell® GT and Sub-Cell Model 96 and 192 systems.
Gels may be cast with a UV-transparent plastic (UVTP) tray
directly on the gel stage of the electrophoresis bases using
the gel casting gates. Gels may also be cast on the removable
UVTP trays with the aid of the gel caster or with standard
laboratory tape.
Casting gels on the base stage with the UVTP tray

1. Level the cell using the leveling bubble provided.
2. Place the UVTP tray on the gel stage.
Note: The Mini-Sub® cell GT requires the 7 × 7 cm UVTP
tray for casting in the GT base. The wide-Mini-Sub cell GT
requires the 15 × 7 cm UVTP tray and the Sub-Cell GT
system requires the 15 × 15 cm UVTP tray for casting in theGT base. Sub-Cell Model 96 system requires the 25 × 10 cm
UVTP tray for casting in the base, Sub-Cell Model 192 system
requires the 25 × 15 cm UVTP tray for casting in the base.
Insert the gel casting guide (table) from page 259(2010/2011 catalog)
3.	Slide the gel casting gates into the slots at opposite endsof the gel stage. Insure that the gates are evenly seated inthe slots and the gates uniformly contact all edges of theUVTP tray. The weight of the gates provides a tight seal toprevent any leakage problems during gel casting*.

* Note: If leakage occurs while pouring the gel on thecasting tray atop the stage, chill the casting gates in thefreezer for 2–3 min. Place the casting gates into the slots
when ready to pour the gel. The chilled casting gates will
prevent the gel solution from leaking out of the tray andinto the chambers.
4.	Place the comb(s) into the appropriate slot(s) of the traysso that the sample wells are near the cathode (black).
DNA samples will migrate toward the anode (red)during electrophoresis.
5.	Prepare the desired concentration and amount of agarose
in 1× electrophoresis buffer. When the agarose solution
has cooled to 50–60°C, pour the molten agarose between
the gates.
Warning: Hot agarose (>60°C) may cause the tray to warp or
craze and will decrease the lifetime of the tray. Warping may
also result in sample wells of uneven depth.
6.	Allow 20–40 min for the gel to solidify atroom temperature.
7.	Carefully remove the comb from the solidified gel.
Remove the gel casting gates.
8.	Submerge the gel beneath 2 to 6 mm of 1× electrophoresis
buffer. Use greater depth overlay (more buffer) withincreasing voltages to prevent pH and heat effects.
Removable tray (UVTP) gel casting using a gel caster or
mini-gel caster1.	Level the gel caster or mini-gel caster using the levelingfeet in the gel caster and the leveling bubble provided.
2.	Disengage and slide the movable wall to the open end of
the gel caster or mini-gel caster by turning and lifting thecam peg upward.
Note: If casting more than one gel with the gel caster,
add the removable gel casting wall to the gel caster.
The removable wall will allow casting using two 15 × 10 cm
trays, four 7 × 10 cm trays or one 15 × 10 cm and one
15 × 15 cm or larger trays.
3.	Place the open edge of the UVTP tray against thefixed wall of the gel caster or mini-gel caster.
4.	Slide the movable wall against the edge of theUVTP tray (Figure 1).

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6206

Lift cam lever up

Movable wall of gel casterEngage and seal(press down and rotate)

Fixed wall ofgel caster

Slidefo rwardFig. 1. Sealing the UVTP tray for gel casting.

5.	To seal the open tray ends, engage the cam peg by
turning and pressing downward simultaneously.
6.	When the cam peg has dropped into the appropriateslot, turn the peg in either direction until resistance is felt.
This action seals the edges of the tray for casting.
7. Place the comb(s) into the appropriate slot(s) of the tray.
8.	Prepare the desired concentration and amount of agarose
in 1× electrophoresis buffer. When the agarose solution
has cooled to 50–60°C pour the molten agarose between
the gates.

Removable tray (UVTP) gel casting using tape

1.	Seal the ends of the UVTP gel tray securely with stripsof standard laboratory tape. Press the tape firmly tothe edges of the gel tray to form a fluid-tight seal.
2.	Level the gel tray on a leveling table or workbench using
the leveling bubble provided with the instrument.
3.	Prepare the desired concentration and amount of agarose
in 1× electrophoresis buffer. When the agarose solution
has cooled to 50–60°C pour the molten agarose into
the gel tray.

Warning: Hot agarose (>60°C) may cause the tray to warp or
craze and will decrease the lifetime of the tray. Warping may
also result in sample wells of uneven depth.

Warning: Hot agarose (>60°C) may cause the tray to warp or
craze and will decrease the lifetime of the tray. Warping may
also result in sample wells of uneven depth.

9.	Allow 20–40 min for the gel to solidify at room temperature.

4. Allow 20–40 min for the gel to solidify at room temperature.

10. Carefully remove the comb from the solidified gel.
11.	Disengage the cam peg by turning and lifting upward.
Slide the movable wall away from the tray. Remove thetray from the gel caster or mini-gel caster.
Note: While the gel is solidifying, a light seal is formed
between the gasket and the gel (especially for low percentage
agarose gels [<0.8%]). Before moving the wall away from thetray, carefully lift the tray on one side to release the seal or use
a spatula to break the seal between the agarose and gasket.

5. Carefully remove the comb from the solidified gel.
6. Remove the tape from the edges of the gel tray.
7.	Place the tray onto the leveled Sub-Cell base so that thesample wells are near the cathode (black). DNA samples
will migrate toward the anode (red) during electrophoresis.
8.	Submerge the gel beneath 2 to 6 mm of 1× electrophoresis
buffer. Use greater depth overlay (more buffer) withincreasing voltages to avoid pH and heat effects.

12.	Place the tray onto the leveled electrophoresis baseso that the sample wells are near the cathode (black).
DNA samples will migrate toward the anode (red)during electrophoresis.
13.	Submerge the gel beneath 2 to 6 mm of 1× electrophoresis
buffer. Use greater depth overlay (more buffer) withincreasing voltages to avoid pH and heat effects.

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Life ScienceGroup

Bulletin 6206 Rev A

US/EG11-0864

1211

Sig 1211