2.10 Pulsed Field Gel Electrophoresis (PFGE)

A. Objectives

1. Prepare agarose plugs containing bacterial cultures for PFGE.
2. Set up and run PFGE to generate DNA fingerprints.
3. Record DNA fingerprints by photographic and digital imaging methods.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

1. Utilize agar cultures to prepare PFGE agarose plugs.
2. Lyse bacterial cells contained in agarose plugs, wash plugs, and perform restriction digestions.
3. Prepare PFGE gel, load plugs, set up and run electrophoresis equipment.
4. Perform staining and documentation of PFGE gel.

C. Questions

1. Describe briefly the preparation of agarose plugs.
2. What are the conditions for plug lysis?
3. Describe the steps needed for plug washing.
4. What restriction enzyme is used for E. coli and Salmonella?
5. What are the two methods for loading plugs into the gel wells?
6. Briefly describe equipment set up for running a PFGE gel.
7. What is the chemical agent used to stain PFGE gels, and what safety precautions are needed to handle this agent?