Pulsed Field Gel Electrophoresis
ProtocolBulletin 6226

Preparing and Running a Gel
This protocol for preparing and running a gel includes
guidelines about casting an agarose gel, gel electrophoresis,
recommendations for avoiding bubbles in the tubing andpreventing curved or slanted lanes, electrophoresis run length,
effect of voltage gradient and using incorrect electrophoresis
conditions, and equipment maintenance.
Casting an Agarose Gel
PurposeAn agarose matrix must be prepared through which theDNA molecules will be separated.
Process■■

 elt a 1% agarose solution completely and allow it toM
equilibrate to 55–60ºC for 15 min before use—	Use 1 g agarose in 100 ml 0.5× TBE buffer
for 10-well gels—	Use 1.5 g agarose in 150 ml 0.5× TBE buffer
for 15-well gels—	Apply short pulses to the solution in a microwave
without boiling over

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 ighten the screws and level the gel form before
Tpouring the gel oad the plug slices on the comb and pour the gel
Laround (preferred) or into the wells of a solidified gel llow the agarose to solidify for ~30 min, but do notAleave it for more than 40 min because the gel will
begin to dehydrate

A video showing how to cast an agarose gel is available at
http://biorad-ads.com/09-0398_CHEF_mapper/09-0398_pop-up.html.

Potential Problems

DNA fragments may not run evenly and spots or debris may
appear on gel images.
Recommendations
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 se high-quality pulsed field gel electrophoresis (PFGE)
U
grade agarose ake sure agarose is completely melted; use a microwave
Mand pulse it in short bursts, but do not boil it overGel Electrophoresis
Recommendations
1.	Check the milliamps (mA) reading at the start of each run.
A reading of 120–165 mA is normal. A high (>165 mA) orlow (<100 mA) reading suggests a problem with the 0.5×
TBE buffer and/or poor water quality.
■■— Buffer dilution or formulation problems

Discard expired agarose eep track of lot numbers and discard deteriorated lots
K
that can be correlated with poor results— Poor-quality water was used to prepare the buffer■■

Remove air bubbles from the gel before it solidifies emove lint, dust, and visible particulates from the gelRbefore it solidifies

Possible reason for a low mA value (<100):— Buffer dilution or formulation problem

Use only glassware, combs, and gel forms that are clean se high-quality water to make the 0.5x TBE buffer used
Ufor the gel and running buffer

Possible reasons for a high mA value (>165):

Note: The mA value will increase during a run (~130–165 mA)
2. Make sure high-quality water is used for the reagents.
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Change filters in the water system regularly■■

Avoid prolonged storage in plastic carboys■■■■

Use sterile, ultrapure (type I or reagent grade)water for all reagents
Nonsterile, ultrapure water can be used for:— 0.5× buffer for 1% agarose gels
— 0.5× electrophoresis running buffer— Destaining gels

A variety of problems may lead to speckles or spots on the gel image.

Electrophoresis instrument screen showing a normal reading, 140 mA.

Electrophoresis instrument screen showing a low reading, 105 mA.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6226

Avoiding Bubbles in the TubingPotential Problems

When Tygon tubing is too long, bubbles can collect inside thetubing. This interferes with steady buffer flow, leading to an
uneven flow rate and temperature fluctuations.
Recommendations■■

 ut the tubing to an appropriate length so it does not hangCat an angle that allows bubbles to collect■■Check that the tubing is relatively straight and free of kinks■■

Check for leaks in the tubing and keep connections tight

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 urn on the pump first before the chiller; ice in the line canTalso affect the circulation and cooling of the buffer onfirm the flow-rate is 1 L/min once a year and adjustC
the pump accordingly onfirm the temperature is 14 ± 2°C once a year andC
replace the probe if necessary

Preventing Curved or Slanted Lanes
Factors contributing to lane curvature:■■

Gel frame was not level when the agarose was poured■■

Gel was not flush against the black frame■■

Electrophoresis chamber was not level■■

Buffer flow was uneven or interrupted■■

Insufficient buffer in the electrophoresis chamber and lines— CHEF Mapper® system requires ~2.2 L of buffer— Older models require ~2.0 L of buffer■■Broken electrode(s)Recommendations
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Balance the electrophoresis chamber before starting a run ake sure the frame is sealed and the screws are tightenedM
before pouring a gel so the agarose does not leak se a leveling platform to ensure the gel is level whenU
the agarose is poured
 nsure the pump is working and the flow rateE
is correct (1 L/min) everse the connections and flush out debris
Rclogging the lines

For more information, watch the instructional video at
http://biorad-ads.com/09-0398_CHEF_mapper/09-0398_pop-up.html.

Cooling module and variable-speed pump for CHEF systems.

Example of a gel with slanted lanes.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6226

Electrophoresis Run Length
The bottom band of the standard (20.5 kb if using PulseNet’s
universal standard strain) should be 1–1.5 cm from the bottomof the gel. If the gel pattern is too short, increase the run time.
This will improve band separation, but the run time should notbe increased so much that the smallest band runs off the gel.

Effect of Voltage Gradient
The voltage gradient also affects band resolution and band
appearance in gels and must be adjusted as needed.

Potential Problems

1. If the run time is too short:■■

Pattern is compressed■■

Decreased resolution of closely migrating bands■■

Normalization of the pattern may be compromised

2. If the run time is too long:■■

Bottom band of the standard runs off the gel■■

Unable to perform normalization

3. Critical factors that affect run times:■■

Composition of 0.5× TBE buffer

3 V/cm for 18 hr— Commercial buffer vs. in-house

The PulseNet universal standard strain, Salmonella enterica, serotype
Braenderup H9812, run with different voltage conditions. A, bands arecompressed; B, appropriate band patterns; C, bands run off the gel.

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Buffer pH■■

Buffer temperature

6 V/cm for 18 hr

9 V/cm for 18 hr— Ambient temperature— Length of tubing■■

Gel concentration

AB

Short
run length

Appropriate
run length

Examples of short and appropriate electrophoresis run lengths.
A, the run time was too short and the doublet was not resolved in the gel;B, the run time was increased and the doublet was resolved in the gel.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6226

Using Incorrect Electrophoresis Conditions
An example of performing gel electrophoresis using thewrong protocol is shown below. In this case, E. coli isolates
were run using Salmonella electrophoresis conditions(2.16–63.80 sec instead of 2.16–54.17 sec). This type of error
is likely to go unnoticed until analysis. It is important to checkfor distortion bars (circled). Organisms run using inappropriate
conditions must be re-run because it is not possible tocorrect for inappropriate conditions in the analysis software
after a run.

Equipment Maintenance
Gel Trap Maintenance

1.	Remove any small pieces of agarose from the top of thegel trap and the floor of the electrophoresis chamber after
the buffer is drained.
2.	Check the gel trap is flush with the bottom of theelectrophoresis chamber to prevent small pieces ofagarose circulating through the lines and clogging thedrain port(s), which may affect buffer circulation.
3.	Replace broken “feet” in the gel trap as needed.
4.	Rinse the chamber and lines with 2 L distilled water toremove residual buffer after a run is complete and thebuffer has been drained.
5.	Spray the equipment down with 70% ethanol andwipe gently.
For more information, view the instructional video at
http://biorad-ads.com/09-0398_CHEF_mapper/09-0398_
pop-up.html.

E. coli isolates run using Salmonella electrophoresis conditions.

Electrophoresis conditions are similar, however dark bars reveal
incorrect band separation and poor normalization.
© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6226Electrophoresis ChamberA

Potential

ProblemsThe chamber and/or tubing can becomecontaminated, affecting the entire gel. Additionally, a single
tube or reagent may be contaminated, affecting only one
or several lanes. In either instance, DNA will be a degradedsmear on a gel.
Recommendations

Keep the chamber clean and dry between runs.
To decontaminate electrophoresis chambers:
1.	Circulate 2 L of 5–10% bleach through the electrophoresis
chamber and tubing for 30 min. Leave chiller off.
2. Drain the bleach solution from the chamber and tubing.
3.	Circulate 2 L distilled water for 15–30 min through thechamberand tubing. Drain and repeat.

B

4. Run the gel with previously tested PFGE plugs.
5. Repeat cleaning if necessary.

Examples showing how contamination of an electrophoresis chamber
impacts gels. A, plug samples were re-run after cleaning the chamberand lines with bleach and rinsing them with water. An additional cycle ofdecontamination is needed and replacing the tubing may also be necessary.
B, contamination of tubes containing plugs of Salmonella enterica, serotype
Braenderup H9812 (—).

Tygon is a trademark of Saint-Gobain Performance Plastics Corporation.

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Bulletin 6226 Rev AUS/EG11-0864

1111

Sig 1211