Lab 2: PROCEDURE Part 2
PROCEDURE: GEL ELECTROPHORESIS
Each pair will prepare one batch of 25 mL agarose and use it to pour one gel. They will share that gel to load their samples.
- Calculate the amount of agarose you will need for 25 mL of a 1% gel. Weigh out the agarose and add it to a 250 mL Erlenmeyer flask. Next, add 25 mL 1× TAE buffer to the flask. (TAE stands for “Tris–Acetate–EDTA.” A recipe for TAE, and for most buffers used in this course, can be found in the Appendix for future reference: Appendix 4: Buffer formulations)
NOTE: The amount of agarose you will be adding to the TAE will not affect the volume enough to be concerned about. However, if you were using 10 g of agarose, you would need to add the solid first and then add just enough TAE to bring the total volume to 25 mL.
NOTE: 1× TAE buffer will also be used as the running buffer for electrophoresis. Therefore, the gel and the running buffer will have the same ionic strength. Failure to use the running buffer to prepare the gel will result in erratic separation of DNA.
The following video demonstrates the gel pouring process in 551 lab. Note that our pouring systems may be different than those you’ve used in other labs.
- Place an inverted 25 mL Erlenmeyer flask or wadded up paper towel in the mouth of the 250 mL flask and heat in a microwave on medium heat until all the agarose has completely dissolved. In our microwave, try heating the gel for 45 seconds, swirl to ensure thorough mixing of the agarose, and then heat for additional 15 second intervals until the solution is clear.
CAUTION: Agarose can become superheated and boil over in the microwave or when swirled. Wear a heat-resistant glove when removing the agarose and swirling heated flasks.
- Allow the transparent agarose solution to cool until you can hold the flask in your hand.
- Meanwhile, place the black rubber bumpers onto the ends of the clear plastic gel casting units. The bumpers are a tight fit so they may be difficult to put onto the casting unit. If you have difficulty sliding the bumpers onto the casting unit, try sliding one side on and then the other. Once you have the bumpers on, place the comb into the unit.
- Once the agarose is cool, add 2.5 μL EtBr (10 mg/mL), and gently swirl to mix. Do not remove the EtBr solution from the designated area. Use the designated pipette.
- Fill a casting unit with the agarose.
Lab Safety Notice
EtBr is a carcinogen! When working with it, please observe the following precautions:
- Work with the provided EtBr stock only in the designated EtBr area.
- Use the designated EtBr pipette. Dispose of pipette tips in the designated EtBr Tip Waste container.
- Wear gloves and eye protection.
- Dispose of EtBr gels in the designated containers.
- Wait about 5–10 min for the gel to solidify; the gel should appear cloudy, not clear.
- Once the gel has solidified, gently pull the comb straight up out of the gel. Be careful not to destroy the wells when you pull out the comb! Next, r