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 H 2 O)  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.