Pulsed Field Gel Electrophoresis
ProtocolBulletin 6224

Plug Preparation
This plug preparation protocol describes how to prepare
intact, chromosome-sized DNA for pulse field gelelectrophoresis (PFGE). It includes guidelines about
preparing the cell suspension, adjusting the cell density,
casting the plug, cell lysis, and washing the plug.

S18 hrS

18 hr

Preparing the Cell Suspension
PurposeA cell suspension is prepared to evenly suspend the cells
within a buffer solution that will maintain the cells in a viablestate until the plugs are cased. The appropriate buffer mayvary from organism to organism. There is no “universal buffer”
for making cell suspensions and preventing cell lysis.
Testing may be required to identify the most appropriate
buffer for the organism, if it is not already known.
Fresh culture

Process

Transfer bacterial cells from the growth media to a cellsuspension buffer.

Old culture

PFGE gel from a fresh culture shows no smearing and all bands aredistinct. Lysed cells are present in 48-hr old cultures (same organism),characterized by smears on the PFGE gel. S, standard.

Potential Problems

Undue stress to bacterial cells prior to casting the plugcan cause cell lysis, leading to DNA degradation, and ischaracterized by “smearing” on the gel.
Recommendations
■■

■■

■■

Grow cultures on nonselective media
 se fresh cultures grown for 14–18 hr at appropriate
Utemperature and conditions
Procedure—	Do not let cultures sit at room temperature formore than 1 hr before making cell suspensions—	Do not store cultures at 4ºC before making cellsuspensions—	Do not use cultures grown for more than 18 hr(contain lysed cells)■■■■

Adjusting the Cell Density
PurposeCell density needs to be adjusted to ensure an optimal
amount of bacterial cells is present in the agarose plug.
Process

As the cells are transferred to the cell suspension buffer,the optical density (OD) or turbidity of the solution should bemeasured. Make adjustments as needed by adding additional
cells or cell suspension buffer until an appropriate value within
the optimized range for that organism is reached.
Potential Problems■■■■

Do not centrifuge or vortex cell suspensions
 o not leave bacterial cell suspensions at roomDtemperature for extended periods of time prior tocasting plugs■■If the cell suspension concentration is too low, not enoughDNA is present and the resulting pattern will be faint anddifficult to analyzeIf the cell suspension concentration is too high, increased
amounts of lytic and restriction enzymes are necessary toachieve complete lysis and DNA digestion, respectively
 s the amount of DNA increases, the band thickness
Aincreases; doublets of bands are difficult to discriminate
and closely migrating bands of similar sizes may appear
as a single bandRecommendations■■■■

 est several different cell suspension concentrations over aTwide range. Even with standardized protocols, the optimal
range may vary slightly across laboratories, depending on
the equipment and reagents used
 ook for the range that results in consistently clean patterns
Lwith limited background and sharp, clear bands

AS

0.1

0.2

0.3

0.35

0.4

0.45

0.5

0.8

S

Casting the PlugPurpose

Encasing the cells prior to cell lysis within an agarose matrix
is necessary to stabilize them and allow for isolation of intact
DNA molecules.
Process

1.	Transfer an aliquot (typically 400 ml) of the cell suspension
to a 1.5 ml centrifuge tube.
2.	Add proteinase K (20 ml of 20 mg/ml stock) to inactivate
damaging nucleases.
3.	Quickly add an equal volume (400 ml) of 1% PFGE-quality
agarose that has been equilibrated to ~54°C.
4. Pipet gently to mix and dispense into plug mold(s).
5. Allow plugs to solidify (~10 min).

Reusable Plug Mold (catalog #170-3622)
BS0.10.2

0.3

0.35

0.4

0.45

0.5

0.8

S50-Well Disposable Plug Mold (catalog #170-3713)

Examples of cell suspensions with cell concentration increasing fromleft to right. Lanes with 0.3–0.5 OD show similar band intensities in cellsuspensions of Escherichia coli (A) and Salmonella (B). S, standard.

© 2014 Bio-Rad Laboratories, Inc.

Bulletin 6224

Potential Problems■■■■

Potential Problems

Agarose that is too hot may damage the cells
 garose thickens and solidifies as it cools, making pipettingAdifficult and leading to an uneven distribution of cells
within plugs

Recommendations
■■

■■

■■

 quilibrate melted agarose to 50–54ºC and keep it warmE
while casting plugs
 imit mechanical force (pipetting) when mixing melted
Lagarose with cell suspensions
 o not re-use plug agarose more than 3 or 4 times;D
repeated heating results in loss of fluid and increases
the agarose concentration
S

1%

1.2%

1.4%

S

1.6%

1.8%

2%

S

If lysis is incomplete, DNA will only be partially digested andfaint bands, smearing, and significant fluorescence in the plug
slice will be observed in the gel.
Note: The amount of material remaining in the wells is agood indicator of the efficiency of cell lysis.
Recommendations■■

 se fresh (not expired) reagents that are kept sterile
Uand uncontaminated■■Decrease cell density■■

Follow correct temperature and shaking recommendations■■

Ensure plugs are completely covered by lysis buffer

Note: When working with a strain for the first time, getting
information from published protocols/articles might help avoid
failed experiments. If you are a pioneer with a strain you canobserve it under the microscope to determine lysis time.
If the strain does not lyse, try a new enzyme or highertemperature. Optimal lysis temperature is usually similar
to optimal temperature for strain growth.
SSS

Gel showing effects of re-using plug agarose. When agarose is reheated,water evaporates and the agarose concentration increases, resulting in plugsthat do not have the expected agarose percentage. Reheating the agarose sixtimes increased the concentration from 1 to 2%. S, standard.

Cell Lysis
Cell lysis is necessary to break the bacterial cell open so
DNA is accessible to enzymes during the subsequent DNA
restriction step. As cells lyse, endogenous endonucleases
are released that must be inactivated to prevent random
DNA degradation.

Examples of incomplete cell lysis. Samples in the indicated lanes were notlysed properly. These lanes display DNA smearing, few visible bands, and cellmaterial in the wells. S, standard.

Process1.	Place 1–3 plugs (same sample) in 5 ml of cell lysis buffer(50 mM Tris:50 mM EDTA, pH 8.0 + 1% sarcosyl +100 µg/ml proteinase K).
2.	Lyse plugs in a shaking (~175 rpm) water bath or
incubator at 54ºC.
Note: Some organisms lyse more quickly or efficiently thanothers; maximal cell lysis occurs within 1–4 hr. Plugs typically
clear as cells lyse.

© 2014 Bio-Rad Laboratories, Inc.

Bulletin 6224

Washing the PlugA

PurposeWashing plugs removes proteinase K, cellular debris, lytic
enzymes, or proteases present during the lysis step that may
interfere with subsequent DNA digestion.
Process ash in 10–15 ml at 50ºC for 10–15 min withWconstant agitation.
■■■■

2 times with sterile clinical laboratory reagent grade water
4 times with TE buffer (10 mM Tris:1 mM EDTA, pH 8.0)

Potential Problems

Inadequate plug washing typically results in incomplete
digestion or smearing.
Recommendations■■■■

Wash plugs 2 more times with TE buffer
Digest and run another plug slice

B

Smearing and incomplete restriction resulting from insufficient washing.
A, the indicated plugs were washed four times with TE buffer; B, the sameplugs were washed six times with TE buffer.

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Life ScienceGroup

Bulletin 6224 Rev B

US/EG14-19791114

Sig 1213