Pulsed Field Gel Electrophoresis
ProtocolBulletin 6225

Restriction Enzyme Digestion
Restriction enzyme digestion is used to prepare DNA foranalysis or other processing steps. Guidelines about preparing
plug slices, selecting enzymes, performing DNA restriction,
troubleshooting restriction steps, and using thiourea to typestrains are included in this protocol.
Preparing Plug Slices
PurposeCut a small portion of the plug for restriction enzyme digestionand electrophoresis. Note that the size and shape of the plug
slice will be reflected in the appearance of the gel pattern.
Process

Use a marked Petri dish, glass slide, or other template
to cut 2 mm–thick plug slices.
Potential Problems■■■■

 lugs that are too small can easily be damaged and result
Pin band distortions
 lugs that are too large can result in thick, indistinct bands
P
that are difficult to analyze

Note: Variation in plug size among samples on the samegel may result in different fluorescence intensities that canbe difficult to appropriately image.

Use a marked Petri dish, glass slide, or other template to cut
2 mm–thick plug slices.

Plug slices
1 mm

123Damaged plug slices
45Recommendation

Only use undamaged plug slices of a consistent size.

Gel showing band distortions. Lanes 4 and 5 were loaded with small plugsthat were physically damaged.

Selecting Enzymes

Performing DNA Restriction

Purpose

PurposeThe optimal restriction enzyme varies across organisms
because the frequency of restriction sites varies. For pulsed
field gel electrophoresis (PFGE), select a rare-cutting
restriction enzyme that produces 15–20 DNA fragments
across a broad range of sizes.

Use a rare-cutting restriction enzyme to cut the genomic
DNA into a small number of fragments that can be resolved
using PFGE.

Process

Process

Prepare a master mix containing:
Restriction enzyme
Restriction buffer recommended by the enzyme vendor■■ Bovine serum albumin (BSA; optional, but recommended)■■ Sterile deionized water■■

Refer to published articles to determine what enzymes havebeen used previously for an organism. If the whole genome
sequence of the organism is available, follow the guidelines
about in silico DNA restriction at http://insilico.ehu.es/.
This site identifies restriction enzymes that produce anappropriate number of DNA fragments and may be usefulfor PFGE analysis. The enzyme(s) should then be evaluatedon a subset of isolates.

■■

Mix thoroughly and aliquot into separate tubes. Check theappropriate temperature for the restriction enzyme being
used and incubate 2–4 hr.
Recommendations■■

AYes
SfilNoApalNoApal■■AsclS

Always use master mixes when preparing enzyme mixtures
 lways use the restriction buffer recommended byAthe vendor■■

Include BSA even with enzymes that do not require it■■

Routinely check water bath temperatures■■

 emove enzyme master mix and incubate digested slices
Rwith 200 ml 0.5× TBE buffer to inactivate enzyme after theincubation step is complete

Evaluation of three restriction enzymes: SfiI, ApaI, and AscI. SfiI is the
optimal enzyme for this sample. S, standard.

© 2014 Bio-Rad Laboratories, Inc.

Bulletin 6225

Troubleshooting Restriction Steps

Recommendations

If incomplete restriction is observed:

Potential Problems

1.	Incomplete DNA restriction appears as “shadow”
or “ghost” bands on gels and can be caused by:■■■■	 — Procedure—	Poor plug quality — proteinase K not removed
from the plug■■

— DNA concentration too high— Enzyme concentration too low■■— Bad lot of enzyme and/or buffer— Enzyme and/or buffer has deteriorated over time

20 U nsure that sufficient units of enzyme are present in theEreaction by increasing the number of units of enzyme
 ake sure the DNA concentration is not too high byM
reducing the initial cell suspension concentration orcutting thinner slices dd high-quality enzyme and buffer that have been
Astored properly and are not expired to the master mix

Tip: To determine whether incomplete restriction is theresult of enzyme or buffer quality, set up another reaction
comparing the current enzyme with a new or previously
tested lot of enzyme.

— Incorrect incubation temperature or buffer
10 U

Wash the plugs 2 additional times with TE buffer

40 U■■Ghostbands

 SA can be added to reaction mixtures even when notB
recommended by the vendor

Note: BSA helps to maintain enzyme stability during thedigestion step. It also minimizes enzyme inactivation due toinhibitors and prevents the enzymes from adhering to the wallof reaction tubes.

Incomplete digestion due to low enzyme concentration results inghost bands. Complete digestion was achieved by increasing the enzyme
concentration to 40 U. U, units.		
SSfilSNotlSS

Lanes 2–4, 6, and 7 show smears caused by expired SfiI ( — ) enzymethat did not cut DNA. The same plugs cut with NotI ( — ) enzyme yielded
distinct bands, indicating that the plugs were usable and smears were due tousing expired enzyme. S, standard.

© 2014 Bio-Rad Laboratories, Inc.

Bulletin 6225

Potential Problems

AAscl

Apal

B

Ascl

Apal2.	When exposed to nonstandard reaction conditions,some restriction enzymes manifest altered specificity
and cleave sequences that are similar but not identical totheir recognition sequence. This phenomenon is called“star activity” (Barany 1988). Star activity results in DNA
degradation, smearing, and few bands.
Conditions that can lead to star activity include:— Prolonged incubation time
— Incorrect buffer or buffer concentration— High (>5% v/v) glycerol concentration—	High enzyme to µg of DNA ratio(that is, too much enzyme)
Note: Star activity can be confused with poor lysis because
the results look similar. Therefore, both steps must be
considered when troubleshooting the appearance ofsmearing with few bands on gels.
Potential Problems

3.	Some strains cannot be cut by certain enzymes
because the enzyme recognition site is not present or
is inaccessible. This results in the appearance of a large
band at the top of the gel with no other bands or DNA
smearing in the lane.
Recommendation

Digest a new plug slice to confirm that there was not an issue
with that individual reaction (that is, the enzyme was omittedor the slice was not treated with the restriction mixture). If thesame result is obtained, it is considered a valid pattern forthat isolate.

Slices from same plugs retested. During overnight incubation of the plug
slices, the enzyme AscI exhibited star activity (A). When slices from the sameplugs were incubated for 2 hr, no star activity was noted (B).

Escherichia coli 026 was digested with BlnI. A single band was confirmed
to be a valid pattern for E. coli 026.
© 2014 Bio-Rad Laboratories, Inc.

Bulletin 6225

Using Thiourea to Type Strains

APurpose

Thiourea is used to type strains that do not yield a pattern,
only a smear (that is, untypeable strains). Thiourea acts as
a reducing agent and neutralizes the oxidizing species thatare generated when running an electrical current throughTris-based buffers. The presence of thiourea during the run
prevents degradation of modified DNA, allowing untypeable
isolates to be typed.
Recommendations

Include a digested plug slice of a previously tested, thioureadependent isolate on the gel as a positive control. Using apositive control verifies that this is a strain problem and nota plug, reagent, or other problem.
■■

■■

■■

If the plugs are deteriorated, DNA will be left in the welland the addition of thiourea will not help dd 50 μM thiourea (873 µl of 10 mg/ml stock) directly toA
the electrophoresis buffer (0.5× TBE buffer) just before
starting the runB se thiorurea only when needed (with known strains orU
suspected serotypes) and not routinely

Caution: Thiourea is a mutagen and proper disposal
is essential. Check with institutional hazardous waste
disposal guidelines.
References
Barany F (1988). The TaqI “star” reaction: Strand preferences revealhydrogen-bond donor and acceptor sites in canonical sequence recognition.
Gene 65, 149–165.
University of the Basque Country. In Silico Simulation of Molecular Biology
Experiments. http://insilico.ehu.es/, accessed November 17, 2014.

Slices from the same plugs were treated without (A) and with (B) urea
and run under the same gel conditions. Lanes 3, 4, 6, 7, 8, and 9 are
thiourea dependent.

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Bulletin 6225 Rev BUS/EG14-1980

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