Materials and Reagents
|For RE digests:
For agarose gel electrophoresis:
* Our lab uses RE reagents from Thermo Fisher Scientific. Buffer R was chosen as the components are compatible with the NdeI/XhoI double digest. The correct buffer can be chosen using the Thermo Fisher Scientific Double Digest Calculator. There are many other companies that sell RE reagents. Please follow the RE buffer recommendations of the company you choose to purchase RE reagents from.
For our specific agarose gel apparatus, we will make a total of 120 mL agarose gel suspension per gel cast. Our agarose gels have 20-lane combs. Please note that this protocol will change depending on your specific agarose gel apparatus. Students will prepare one 120 mL agarose gel during the 30-minute restriction enzyme digest incubation.
1. Weigh out the appropriate mass of agarose into a 500 mL Erlenmeyer flask and add 120 mL of 1X TAE electrophoresis buffer.
Empty 2L bottles and a small aliquot of 50 X TAE will be provided. Using these reagents designated students should make 2 L of 1 X TAE. Please note it is not necessary for every student to make this solution; this will ultimately be dictated by class size. Prior to the start of the lab, each student is responsible for calculating the amount of 50 X TAE and water required to make a 2 L bottle of 1 X TAE. Please complete this calculation in your lab notebook prior to the lab time.
2. To mix, gently swirl the contents of the flask. Please stopper the top of the Erlenmeyer flask with a wad of paper towels so as to avoid spillage and evaporation during microwaving. Please ask your instructor for help with this step.
3. To dissolve the agarose, heat the 120 mL agarose solution in a microwave for about 2 minutes on high (this step requires training and supervision from your instructor and proper safety precautions: face shield). Point the open mouth of the flask away from your face and gently swirl the solution to ensure proper mixing (use oven gloves to handle flask). If the agarose is not completely dissolved heat again for 30 seconds on high. Monitor your flask when heating to avoid the solution boiling over. Once the agarose is dissolved wait for 30 seconds for the solution to cool and use oven gloves to remove the hot agarose from the microwave. Point the open mouth of the flask away from your face and gently swirl the solution to ensure proper mixing. Carefully take the flask back to your bench and alert the students in your team that the flask contains hot liquid.
4. Allow the agarose to cool to 60 °C. In the meantime, set up the gel apparatus with assistance and training from your instructor.
5. Once the solution has reached ~60 °C, ask your instructor to add 3 µL of GelRed. Pour the agarose gel solution into the gel casting tray. Remove any bubbles (a small pipette tip is good for this) and place the comb into position (20 lanes/ comb). The gel will take 20-30 minutes to solidify. Once the gel is solidified remove the tape from both ends of the casting tray before placing it in the running apparatus (please make sure that gel does not slide out of the casting tray when you are removing the tape). Add 1 X TAE buffer to submerge the gel (You will also need ~ 600 mL of 1X TAE to submerge the agarose gel once solidified). Your lab instructor can help you with this step.