19.1: Instructor Guidelines
- Page ID
- 118042
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Students are introduced to restriction enzymes and gel electrophoresis during this molecular biology lab. This is a 2-day lab, as students will run the gel during day one and view their results during the following lab. Students will use three different restriction enzymes to digest lambda DNA, and then separate their fragments using gel electrophoresis. Students will also create a standard curve using the HindIII digest and use this curve to determine the sizes of the unknown fragments generated by the EcoRI and PstI digests.
Tasks
Students will complete the following during this lab:
- Use three restriction endonucleases, PstI, EcoRI, and HindIII, to digest lambda bacteriophage DNA.
- Cast an agarose gel with DNA wells, set up an electrophoresis chamber, and load DNA samples into the gel.
- Stain and destain an agarose gel in order to identify DNA fragments.
- Create and use a standard curve to identify the sizes of unknown DNA fragments.
Day one of this lab is a long, intensive 3-hour lab that may require the instructor or lab technician to take additional time to stain or destain the gels after the lab in order to have the gels ready to view and analyze during the following lab. Although a pre-lab reading quiz is recommended, waiting until the middle of the lab session (e.g. during incubation or while the gels are solidifying) to complete the quiz may work best to ensure all of the steps can be completed during the lab period.
All of the students in each group should practice using the micropipetters several times before beginning the experiment. All students should also practice loading the practice gels with the practice loading buffer solution.
The restriction enzymes should be kept at the instructor's desk so that the instructor can ensure the proper volumes are added to each microcentrifuge tube. Casting trays will need to be taped on the top and bottom edges to prevent the agarose from leaking. Remind students that the height of the agarose should be just below the edge of the comb teeth to create proper wells for loading.
Once the agarose has set, flooding the wells with dI water will help students remove the combs without breaking the wells.
Once the power supplies have been turned on, ask students to check for small bubbles to ensure the chamber is working properly. Remind students to check their gels 5 minutes after starting to ensure the bands are running in the correct direction. During this time, each group of students should prepare a plastic Tupperware storage container (or equivalent) by labeling the lid with tape, and pouring enough methylene blue into the box to cover their gel. Gels will be transferred into the storage container to be stored in the refrigerator overnight. 24-hours later, the gels should be destained by removing the methylene blue and replacing with dI water for 24- hours. If more than 48-hours will pass before the next lab session, gels should be removed from the dI water, wrapped in plastic wrap to prevent drying, and stored in the storage container in the refrigerator.