Teacher's Guide: Techniques Lab C-1
Casting and Loading an Agarose Gel
Time of lab
Preparation before lab
- One FULL period. Explain lab procedures the day before you do the lab.
- Mix one-two batches of 0.8% agarose in 1X TAE buffer (not water!).
- For example, 400 mL of 0.8 % agarose would be enough for 15-20 teams, each using 20-25 mL. To make this, mix 3.2 g of agarose power in 400 mL of 1X TAE buffer. SEE TECH TIPS BELOW.
- If you are using agarose sent from previous schools, keep an eye out for 2 possible problems:
- Wrong concentration agarose. If much less than 0.6%, gels will be flimsy and break easily. If much greater than 1%, dyes will migrate more slowly and separate very little in the time allotted.
- Improper buffer concentration. Gels made in water will solidify clearer than gels made with buffer. Dyes may move into adjacent wells. Dyes will be smeary if run 20+ minutes. Check pH if you suspect water. Gels made in higher than 1X buffer will heat up more quickly and dyes will be smeary. Check current for clue about buffer concentration.
- Check that loading dye has been aliquoted. 100 ÁL of loading dye per team will be enough. The loading dye contains 0.25% xylene cyanole (XC) and 0.25% bromphenol blue (BPB) in 50% glycerol, 10 mM Tris, 1 mM EDTA, pH 8. XC is a negatively charged dye that migrates slower than most DNA; BPB is a negatively charged dye that migrates faster than the DNA sizes we use. Glycerol is used to increase the density of the loaded sample so that the sample sinks down into the wells.
- RE-USE the agarose throughout the day. Collect the previous class' gels in a 800-1000 mL beaker and liquefy before dispensing to next class. (Don't be surprised if the agarose begins to turn blue.)
- Carboys with 1X TAE should be set out in the classroom. To expedite dispensing of buffer, provide several beakers near the carboys.
- Only the first class of the day needs to fill gel boxes. 1 X TAE will "last" for an entire day; it should NOT be discarded at the end of each period! Gently slosh the boxes to mix the ions...
- Nothing needs to be sterile for this lab.
What to demonstrate/explain
- Show how to raise or lower, and secure, the "gates" on the ends of the casting trays.
Unless you tell them not to, students may remove the screws entirely. They need only be loosened. Students might also overtighten the screws. Screws should be adjusted so that the gates are snug with the edges of the tray, yet the gates can be moved up and down with reasonable effort. Once the tension on the gates has been properly adjusted, it is not usually necessary to loosen and retighten the screws with each use.
- Review how to properly slide off gel box lid. DO NOT PULL ON WIRES!!!
- Review how to use pipet for tiny volumes of viscous solutions (Lab A2).
- Demonstrate how to load a well without "puncturing" the bottom of the gel with the pipet tip.
- Careful -- loading dye tubes "splatter" as they are opened. The dye stains hands and clothes.
- KEEP TRACK OF COMBS! They cost $10.00 each!!! (You might want to color the edges with red or green marker to make the combs more visible, especially on white paper towels!)
- Dissolve (or re-liquefy) agarose by microwaving. It takes only 5-6 min. on MEDIUM to liquefy a 500-mL bottle of agarose. THE LID MUST BE LOOSE. After heating, do not swirl too vigorously, or superheated agarose can bubble out of the bottle.
- HOLD melted agarose in a 65 degrees C water bath for use during the period. Students dispense what they need (20-25 mL), insuring that the agarose will not be so hot that it warps the casting trays as it is poured.
- Our choice of 20-25 mL is a trade-off between gel running time (thinner runs faster) and gel durability (thicker gels are easier to handle without tearing).
- A common error: wells of gel at wrong end of gel box.
Loading dye will migrate toward the positive end of the box (anode),
so the wells of gel should be at the cathode (negative; black) end of the gel box.
You can make a mixture of 4 dyes: bromphenol blue, xylene cyanole, phenol red and Orange G. By varying the combinations, you could have students determine which samples have the same dyes in them.
Another extension or modification of this activity is to put the wells in the center of the gel and have students load various dyes, some with positive charge, some with negative charge, and observe the dye migration. See Lab C-2.
- At the end of the day, after all classes have completed the lab, the 1X TAE and the blue agarose can be discarded.