Pulsed Field Gel Electrophoresis
ProtocolBulletin 6223

General Considerations for Troubleshooting PFGE Gels
Pulsed field gel electrophoresis (PFGE) has enabled progressin cancer research, food safety, public health, quality
control, and genome mapping. It is widely used in molecularepidemiology for strain typing and it has been adopted byPulseNet, a global network of health and food regulatory
agency laboratories coordinated by the Centers for Disease
Control and Prevention (CDC).
After obtaining a poor PFGE result, troubleshooting isnecessary to resolve the problem and ultimately achieve
high-quality PFGE gels again. High-quality, reproducible PFGE
data allow laboratories to exchange and compare accurate
information. For PulseNet member laboratories, PFGE data

help prevent disease through real-time subtyping of foodborne
pathogens and cluster detection followed by a swift andfocused public health response.
Major procedural steps of PFGE include:
Preparation of cell suspension
Preparation of agarose plugs■■ Lysis of cells in agarose plugs■■ Washing of agarose plugs■■ Restriction digestion of DNA trapped in agarose plugs■■ Gel electrophoresis of digested DNA■■ Documentation of PFGE gel■■■■

PulseNet Standardized Protocol for PFGE

All images are courtesy of Kara Cooper and Molly Freeman, Centers for Disease Control and Prevention, Atlanta, GA.

 roubleshooting PFGE gels can be very complex
Tand depends on a variety of factors:■■

Become familiar with the purpose of each step within theprotocol and the potential impact of failure within each step tofacilitate the review of PFGE gels. When reviewing PFGE gels:■■■■

Individual laboratory skills and techniqueLaboratory setup and equipment■■ Reagents■■ Water quality■■ Interruptions and distractions during experiment— Equipment— Reagents

Many problems with poor PFGE results can be solved
by correcting poor laboratory practices:■■

Measure weights and volumes accurately■■

Make accurate calculations■■■■

 onfirm all instruments and equipments areC
working properly heck reagents for precipitation, discoloration,
C
cloudiness, and expiration date■■

Use reagents from commercial suppliers■■

Wear gloves throughout the entire procedure■■■■— Personnel■■■■

 eview all steps of the protocol and consider if the failureR
could be the result of problems within one of those steps
 xamine all areas of the PFGE gels to identify the ones that
Eare unusual because those areas may indicate the root
cause of the failureSSS ake sure glassware is clean without any residualM
detergents or other debris, which can interfere with plug
preparation and lysis or cause specks in the background o not reuse plasticware unless it can be washed
Dand rinsed properly■■

Do not overheat agarose solutions■■

Mix enzyme and buffer mixtures well■■ etermine if there have been any changes sinceD
the last good gel in:— Procedure se appropriate reagents, enzyme, and electrophoresis
Uconditions for each organism

Example of a PFGE gel. Colored brackets indicate different areas of thegel that should be analyzed during troubleshooting. S, standard.

Bio-RadLaboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00
France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300
Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04
Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55
Taiwan 886 2 2578 7189 Thailand 800 88 22 88 United Kingdom 020 8328 2000

Life ScienceGroup

Bulletin 6223 Rev A

US/EG

11-08641111

Sig 1211