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