Casting an Agarose Gel

1. Casting an Agarose Gel Tricks of the Trade

2. Setting up the Casting Tray • Put the casting tray on level, non-movable surface in a place where it won’t be disturbed. • Seal off ends of tray (tape, dams). • Insert comb at one end, seat completely down into the slot. • Measure the volume of tray (usually ~25mL)

3. Measuring Agarose • Comes as solid, used as % solution: example: 1% agarose = 1 g/100 mL. 0.8% = 0.8 g/100 mL • Weigh out and put into container that is at least 3-4 times the volume that you are making up.(erlenmeyer flask)

4. Dissolving Agarose-Microwave method • Add BUFFER to pre-weighed agarose in flask. (not water) • Swirl, stir well to suspend. • Cover outlet of flask with plastic wrap but VENT always – can explode with overheating in microwave! • Heat on high for 1-2 minutes – check frequently • Check carefully that it is dissolved – just before dissolving, you can see crescent-shaped grains.

5. Dissolving Agarose –Heating • Add BUFFER to pre-weighed agarose in flask. (not H2O) • Swirl, stir well to suspend. • Heat in boiling water bath (double boiler fashion) or on hot plate till dissolved – about 10 minutes. • Check for complete dissolution

6. Pouring Agarose • When melted agarose in buffer has cooled to about 50-60 degrees C., pour into casting tray to depth of about 5 mm. • Agarose should reach 1/3 height of comb teeth. • Bubbles are troubles: use pipet tip to move large bubbles/debris to side. • Gel becomes cloudy as it solidifies:15-20 minutes. Do not disturb while solidifying.

7. Options • You can store solidified agarose at room temperature and remelt at later date. • Watch for too much evaporation – add buffer if needed. • You can store poured gels in refrigerator: moisten with buffer and wrap with plastic wrap or put in ziploc bag.

8. Using the Gel • Unseal ends of casting tray. • Place tray into gel box so wells are at negative (black) electrode end. • Fill box with buffer: enough to just cover top of gel.(~110 mL) • can reuse buffer a few times. • Pull comb straight up and out – do not rock or wiggle.

9. Loading the Gel • Put gel box on dark surface (dark paper) to make wells easy to see. • Hold tip over well, barely pierce buffer surface, slowly express sample. • Do NOT insert tip directly into well. • Sucrose or glycerol in loading dye makes sample sink into well.