Presentation on theme: "Casting an Agarose Gel Tricks of the Trade. Setting up the Casting Tray Put the casting tray on level, non- movable surface in a place where it won’t."— Presentation transcript:
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)
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)
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.
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
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.
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.
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.
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.