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, remove the bumpers by gently twisting them off. After removing the comb and the bumpers, place the casting unit into the running box. Two casting units can fit into the same running box, so you can share with another pair.
NOTE: Whether you place your wells on the right or the left will depend on which direction you expect your samples to move and which electrode you use as the cathode or anode. Make sure your gel is facing the correct direction or your DNA will run into the buffer and not the gel!
- Add approximately 350 mL of 1× TAE buffer to the running unit so that the buffer just covers the gel. Make sure that both gels are covered with buffer. If there is used TAE buffer available, please use that.
- For each of your PCRs, place 5 μL H2O into a new microfuge tube, then add 5 μL of your PCR product. Be sure you pipette the liquid all the way down into the water in the tube. Add 2 μL of 6× loading dye and mix by pipetting up and down.
- Load 5 μL of the 1 kb ladder onto the gel. Load all 12 μL of your PCR+loading dye samples onto the gel in separate wells.
NOTE: Be sure to write down the order in which you load your samples and the volume that you loaded. You will need this information and it will be required for your lab report.
- Once all of the samples have been loaded into both gels, gently place the cover on the running box (be careful not to disrupt your samples). Attach leads to power supply. Set the voltage at 150 V and press the run button.
NOTE: Check that your anode and cathode are attached appropriately to run your DNA in the desired direction.
NOTE: Give your gel a few minutes to get started and then make sure it is running as expected. There should be small bubbles moving up from each electrode, and you should be able to see your loading dye begin to migrate.
- Continue electrophoresis until the bromphenol blue (the leading, darker dye) has migrated to about 1/2 of the way to the end of the gel.
- Take a picture of your gel using the gel doc, with help from the teaching staff.
- Please pour your TAE buffer into the “Used TAE” carboy so it can be re-used.