Bio-Rad ReadySub-Cell GT Cells - Manual (Page 9)

Table 2.1 Gel concentration required for DNA separation 1—2
Gel Concentration (%) DNA Size
0.50 1—30 Kb
0.75 800 bp–10 Kb
1.00 500 bp–10 Kb
1.25 400 bp–7 Kb
1.50 200 bp–3 Kb
2.00* 100 bp–2.5 Kb
3.00* 40 bp–2 Kb
4.00** 10–400 bp
* Sieving agarose such as Certified PCR agarose
** Sieving agarose such as Certified low range ultra agarose
Table 2.2 Gel volume requirements
Gel Size (thickness)
0.25 cm 0.5 cm 0.75 cm 1.0 cm
Base
7 x 7 cm
10 ml 20 ml 30 ml 40 ml
15 x 7 cm 20 ml 40 ml 60 ml 80 ml
15 x 15 cm 50 ml 100 ml 150 ml 200 ml
Tray
7 x 7 cm
10 ml 20 ml 30 ml 40 ml
7 x 10 cm 15 ml 30 ml 45 ml 60 ml
15 x 7 cm 20 ml 40 ml 60 ml 80 ml
15 x 10 cm 30 ml 60 ml 90 ml 120 ml
15 x 15 cm 50 ml 100 ml 150 ml 200 ml
15 x 20 cm 70 ml 140 ml 210 ml 280 ml
15 x 25 cm 90 ml 180 ml 270 ml 360 ml
2. Add the agarose to a suitable container (e.g., 250 ml Erlenmeyer flask, Wheaton bottle, etc.). Add
the appropriate amount of 1x electrophoresis buffer (see Section 3, Gel and Electrophoresis Reagent
Preparation, for electrophoresis buffer preparation) and swirl to suspend the agarose powder in the
buffer. If using an Erlenmeyer flask, invert a 25 ml Erlenmeyer flask into the open end of the 250 ml
Erlenmeyer flask containing the agarose. The small flask acts as a reflux chamber, allowing long or
vigorous boiling without much evaporation.
Note: A mark can be put on the lower flask at the same level as the liquid. If evaporation occurs, water
can be added to bring the liquid back to the original starting level.
3. The agarose can be melted by boiling on a magnetic hot plate (Step 4a) or in a microwave oven (Step 4b).
Caution: Always wear protective gloves, safety glasses, and a lab coat while preparing and casting
agarose gels. The vessels containing hot agarose can cause severe burns if allowed to contact skin.
Additionally, molten agarose can boil over when swirled.
Magnetic hot plate method
4a. Add a stir bar to the undissolved agarose solution. Heat the solution to boiling while stirring on a
magnetic hot plate. Bubbles or foam should disrupt before rising to the neck of the flask.
Microwave oven method
4b. Place the gel solution into the microwave. Using a low to medium setting, set the timer for a minimum of
5 minutes, stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the
undissolved agarose. This technique is the fastest and safest way to dissolve agarose.
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