Gel Electrophoresis

1. Gel Electrophoresis Teacher Instructions VWR Set Up 12 groups Mira Costa kit

2. 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)

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

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

5. 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 REDO

6. Prepare Agar for Gels • Measure agar powder into the 15 ml agar measuring tube – tap firmly to settle to 4ml mark • Add measured agar powder to agar mixing bottle • You’ll need to make 1 bottle per class

7. 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

8. 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 immediately once a good boil starts

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

10. Prepare Agar for Gels • Check that agar has fully dissolved or, if re-melting solidified agar, that it has all melted back into solution • Add water (DI or tap) as needed to compensate for evaporation to bring volume back to 300ml

11. Prepare Gel Casting Trays • Allow Agar to cool until you can just stand holding the bottle with your bare hand • While Agar is cooling assemble gel casting trays • 3 casting trays per class

12. Pour Gels • Carefully pour 100 ml warm agar solution into each assembled gel casting trays (make sure agar is still fully melted) • Place 2 combs into appropriate slots on each tray

13. How to store prepared gels • After gels have solidified, remove the combs carefully lift out gel trays • There will be some gel ‘film’ on the bottom • Carefully slide gel out of the tray onto a “patty pac” paper • 4 gels per paper • Wipe excess gel off casting tray and reassemble for next pour

14. How to store prepared gels • Place papers with 4 gels in a gel storage container • Make sure paper edges are free

15. How to store prepared gels • Stack second and third (etc) layer of gels in storage container and place container in fridge for up to a week • Kit design is for 2 classes per container • If storing more per container, place 6 gels per layer to distribute weight on bottom level

16. 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 • One layer at a time • Carefully use spatula to lift each gel from paper and replace into gel tray

17. Setting up prepared gels for class • Place each gel onto flat tray for each student group • Try to keep gels and trays low and level to prevent accidental tearing of the gel

18. Running gels • Prepare 1X TAE Buffer solution for running gels: • Measure 16ml of 50X TAE Buffer stock solution into the 50ml conical TAE Buffer measuring tube • Pour the 16ml of 50X TAE Buffer stock solution into the 2L TAE Buffer mixing bottle • Fill 2L TAE Buffer mixing bottle to 800ml line with water (tap or distilled)

19. Running gels • Pour ~ 250ml of mixed 1X TAE running buffer into each electrophoresis box

20. Running gels • After students have loaded their gels carefully place each gel tray 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

21. Running gels • Top off each box as needed with TAE to bring level to just cover gels • Connect power supplies • Place lids on boxes

22. Running gels • Turn box power on (switch in back) • Use arrows: • Adjust voltage to 120 • Adjust time to 20 min • Can increase voltage to 150 and decrease time to 15 if needed • Press power • If “lid” alarm sounds, turn off power, adjust lid, start again • May need to place large rubber band around box to ensure magnetic connection

23. After Gel Run • When the run is complete (colors have separated) turn off the power • Remove the lids from the electrophoresis boxes

24. After Gel Run • Carefully remove gel trays from the box and depending on time: • Carefully slide each gel from gel tray onto a flat tray and give back to groups to analyze • OR carefully slide each gel from gel tray and place each 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!!

25. 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

26. 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