Gel Electrophoresis

Gel Electrophoresis Teacher Instructions BioRad Set Up 12 groups Grossmont Kit

Timeline • Prepare Gels: Up to a week in advance • Class lab: 1-3 days • Teach students to pipette • Load and run gels • Teach electrophoresis theory • Analyze gels • Gels must be analyzed no later than next day after running (stored in refrigerator overnight)

Prepare 1X TAE Buffer for making gels • Measure 36ml of 50X TAE Buffer stock solution into the 50ml conical TAE Buffer measuring tube

Prepare 1X TAE Buffer for making gels • Pour the 36ml of 50X TAE Buffer stock solution into the 2L TAE Buffer mixing bottle

Prepare 1X TAE Buffer for making gels • Fill 2L TAE Buffer mixing bottle to the 1800ml line with water (tap or distilled) • You might need to repeat this as necessary for your number of classes – this bottle should prepare enough 1X TAE Buffer for 6 classes worth of gels Increase level to 1800ml

Prepare Agar for Gels • Measure agar powder into the weighing dish on the scale (tare first!) to 1.50 grams (ok if within 1.47-1.53) • Add measured agar powder to agar mixing bottle • You’ll need to make 1 bottle per class

Prepare Agar for Gels • Fill each bottle to the 300ml mark with your prepared 1X TAE Buffer solution • Bottles have been pre-checked for calibration • Cap tightly and shake to mix

Prepare Agar for Gels • Loosen caps slightly and place bottles in your microwave • Set microwave for 1-2 minutes per bottle (less is better - you can always add more time!) • Allow agar solution to come to a boil - stop microwave once a good boil starts

Prepare gels • Carefully remove the HOT bottle from the microwave and swirl - be careful of steam escaping from the loose caps!

Prepare Agar for Gels • Check that agar has fully dissolved or, if re-melting solidified agar, that it has all melted back into solution • Allow Agar to cool until you can just stand holding the bottle with your bare hand

Prepare Gel Casting Trays • Place 2 gel trays into casting units • Place 2 combs into slots on each tray (top and bottom)

Pour Gels • Carefully pour warm agar solution into the assembled gel casting trays (make sure agar is still fully melted) • Fill each tray approximately .75cm • 1 bottle should fill 6 trays

How to store prepared gels • After gels have solidified, remove the combs • Carefully slide gel out of the tray onto a “patty pac” paper • 1 gel per paper

How to store prepared gels • Place 2 papers with 1 gel each side by side in a gel storage container • Make sure paper edges are free

How to store prepared gels • Stack second layer of gels in storage container and place container in fridge for up to a week • 1 container per class! • (4 full size gels)

Setting up prepared gels for class • When you are ready to have students use gels simply carefully lift paper with gels out of the storage container • Carefully use spatula to cut each gel in half (~1cm above middle wells)

Setting up prepared gels for class • Place each gel half onto flat tray for each student group • Put a patty pac under if too hard to see wells • Try to keep gels and trays low and level to prevent accidental tearing of the gel

Running gels • Prepare 1X TAE Buffer solution as before: • Pour the 36ml of 50X TAE Buffer stock solution into the 2L TAE Buffer mixing bottle • Fill 2L TAE Buffer mixing bottle to the 1800ml line with water (tap or distilled) • Pour 1X TAE into electrophoresis boxes stopping about 1cm below MAX line

Running gels • After students have loaded their gels carefully slide each gel half back into each gel tray (in same direction) and place into the electrophoresis boxes • Keep track of which groups’ gels are where! • Make sure the well sides of the gels are on the BLACK electrode side • Back to Black, RUN to RED

Running gels • Top off each box as needed with TAE to bring level to MAX line • Place lid on each electrophoresis box making sure that the black electrode is at the well end of the gels

Running gels • Connect the electrodes from each box to the power supply and turn on the power by the switch in the back • Make sure the power supply is set on volts and adjust the voltage to 200* • When you are ready to start the run simply press the button in the center • Watch for bubbles in the electrophoresis boxes! *may need to lower voltage

After Gel Run • When the run is complete (colors have separated) turn off the power by pressing the button on the left • Remove the lids from the electrophoresis boxes

After Gel Run • Carefully remove gel trays from the box and depending on time: • Place each ½ gel on a flat tray on a patty pac and give back to groups to analyze • OR place each ½ gel on a labeled patty pac and store back in storage container in refrigerator until next class meeting and then distribute on flat trays • WARNING - WET GELS ARE VERY SLIPPERY!!

Next period and so on… • You can prepare per 2 gels for distribution while per 1 gels are running and so on… • Running TAE buffer is good for all classes – no need to replace unless it gets too hot

Clean up • At end of day used buffer can just be flushed down sink • Rinse boxes and let air dry • Used gels can be placed in general trash

Gel Electrophoresis