Roche Applied Science
Restriction Enzymes
FAQS and Ordering Guide
A Tradition of Premium Quality
and Scientific Support
Roche Applied Science
Restriction Enzymes
FAQS and Ordering Guide
2
Restriction Enzymes from Roche Applied Science ­
A Tradition of Premium Quality and Scientific Support
Roche Applied Science introduced the first restriction enzymes in 1976. Since then
many researchers have chosen to apply our enzymes in their everyday work and have
relied on the quality and consistency we provide. The key to our success lies in:
Guaranteed stability
Use an enzyme whose purity and full activity are guaranteed for up to 24 months.
Roche Applied Science gives you the security of a expiration date for each enzyme ­ and
guarantees 100% activity through that date.
Selection
With more than 115 restriction enzymes now available, Roche Applied Science provides
restriction enzymes that span a wide range of recognition sequences, including some
which cannot be ordered from any other supplier. Choices range from rare cutters for
genomic mapping to standard enzymes thus reagents and kits for upstream and down-
stream applications are offered by RAS.
Purity and function testing
Obtain the results you expect by using enzymes tested for endonuclease, exonuclease
and phosphatase activity (Figure 1), as well as with the ability to recut DNA after sub-
sequent ligation.
Figure 1: Absence of exonuclease contamination. One
microgram of DNA was incubated for 4 hours with various
amounts of Sma I from different suppliers to test for exonu-
clease contamination. Only the restriction enzyme from
Roche Applied Science (RAS) showed absolutely no exonu-
clease contamination. Exonuclease activity may lead to the
subsequent cloning of incomplete DNA.
Guaranteed cutting activity
Achieve complete digestion with just one unit of a
Roche Applied Science restriction enzyme (Figure 2).
Use an enzyme whose activity is 100% guaranteed
when used in the appropriate SuRE/Cut Buffer.
Figure 2: Complete digestion in one hour. pUCBM21 DNA was cut
with 1 unit BamH I from different suppliers (each enzyme in its compli-
mentary buffer) for 1 hour. Only the restriction enzyme from Roche
Applied Science (RAS) completely cut the DNA.
RAS A
CB
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Restriction Enzymes from Roche Applied Science ­
A Tradition of Premium Quality and Scientific Support
Buffers
Perform restriction digests with more than 115 restriction enzymes, using only five
optimized SuRE/Cut Buffers. The SuRE/Cut Buffer system takes the guesswork out of
double digests. Roche Applied Science includes a complementary vial of SuRE/Cut
Buffer with each restriction enzyme, and guarantees 100% activity with each corre-
sponding enzyme.
Convenient dual concentration
Choose from the most extensive line of high and low concentration restriction enzymes
available.
Service
Expect customer service that is committed to filling and shipping each order the same
day it's received; plus technical service scientists with the expertise to answer questions
on any product in Roche Applied Science's complete product line.
Beside our tradition of premium quality, Roche Applied Science is also known as the
information provider in terms of comprehensive reference material and a committed
product support. To follow our reputation, we have prepared Frequently Asked
Questions in short FAQs about restriction enzymes. They also include questions
focused on troubleshooting.
The FAQs included in this short manual are organized in the following way:
Basic Information
Information provided by Roche Applied Science
Standard Digest
Double Digest
Genomic Digest
DNA Methylation
Troubleshooting
4
Restriction Enzymes from Roche Applied Science ­
A Tradition of Premium Quality and Scientific Support
Reference materials available are,
b The additional information available
in the Biochemicals Catalog.
b The comprehensive Restriction
Enzyme Poster well known as the
information source for commercially
available restriction enzymes and
their recognition sequence.
b The additional information available
in Lab FAQS-Find a Quick Solution
(2nd
edition).
b The handy Laminated Buffer Chart
containing all the information need-
ed for quick selection of optimal
buffer and reaction conditions for
your restriction enzymes.
b The convenient RE Finder Program located on our Bench Mate website,
http://www.roche-applied-science.com/benchmate/ helps you to identify the
enzymes that will cut your DNA sequence, and displays the names and recognition
sequences of enzymes and isoschizomers. Thus detailed information (pack inserts)
of these respective restriction enzymes are linked to your search result.
b Also note the Biochemica Newsletter articles available (also on our website):
Restriction Enzymes from Roche Applied Science ­ A Tradition of Premium
Quality
Roche Applied Science Biochemica 2002; 4:31
Restriction Enzymes Carrying an ATG Sequence in the Recognition Site
Roche Applied Science Biochemica 2002; 4:32
Activity of Restriction Enzymes in a PCR Mix
Roche Applied Science Biochemica 1997; 3:25
b And last but not least, visit our Online Technical Support at
http://www.roche-applied-science.com/support
for further product information.
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Content
Frequently Asked Questions - Topics Page
Basic Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Information provided by Roche Applied Science . . . . . . 11
Standard Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Double Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Genomic Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
DNA Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
6
Basic Information
What are restriction enzymes, and what is their biological role?
Restriction endonucleases are endo-deoxyribonucleases that recognize specific DNA
sequences, and digest double-stranded DNA by cleaving two phosphodiester bonds
(one within each strand of the duplex DNA).
Restriction enzymes form part of the restriction-modification system of bacterial cells
that provides protection against invasion of the cell by foreign DNA - in particular,
bacteriophage DNA. Protection against self-digestion is achieved by the presence of
specific DNA methyltransferases, which transfer methyl groups to adenine or cytosine
residues to produce N6
-methyladenine or 5-methylcytosine. Unmodified foreign DNA
entering the cell is degraded by the host restriction-modification system (see figure
below).
What types of restriction enzymes exist, and which are used in
molecular biology applications?
All restriction endonucleases and their corresponding DNA modification methyltrans-
ferases have been classified into three classes - I, II, and III - according to their gene and
protein structure, cofactor dependence, and specificity of binding and cleavage.
Class I enzymes exhibit both restriction and DNA modification activities that are
located on different subunits of multifunctional enzyme complexes. They require Mg2+
ions, ATP, and S-adenosylmethionine (SAM) as cofactors. These enzymes cleave DNA
at nonspecific sites, usually 100 to 1000 bp downstream of their recognition sequence.
continued on next page
1
EcoR I MEco RT
Restriction Modification
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
AATTC
G
EcoR I
Me
Me
3o
5o
5o
5o
5o
5o
+
5o
5o 5o
3o
3o
3o
3o
3o
3o
3o
2
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Basic Information
What types of restriction enzymes exist, and which are used in
molecular biology applications? (continued)
Class II restriction enzymes and their corresponding modification methyltransferases
act as separate proteins. The enzymes in this class are site-specific, and hydrolyze specific
phosphodiester bonds in both strands of the DNA, within or in close proximity to their
recognition sequence. They require only Mg2+
ions as cofactors.
Class III enzymes, like Class I enzymes, combine restriction and modification activi-
ties in a single enzyme complex composed of different subunits. These enzymes re-
cognize specific sequences, but cleave 25 to 27 base pairs in a 3´ direction outside of the
recognition sequence. They require Mg2+
ions for activity, but lack both the ATPase
activity of Class I enzymes and their absolute requirement for SAM.
Due to their absolute sequence specificity, Class II restriction endonucleases are gen-
erally used as key reagents for a variety of applications in molecular biology and
recombinant DNA techniques, including genomic mapping, restriction fragment
length polymorphism (RFLP) analysis, DNA sequencing, and cloning. In this respect,
they can be considered to be the "work horses" of molecular biology.
Which cleavage sites are recognized by Class II restriction
endonucleases?
Class II restriction endonucleases recognize short nucleotide sequences, and cleave
double-stranded DNA at specific sites within or adjacent to those sequences. The length
of these recognition sequences varies considerably. For example, the enzyme Not I rec-
ognizes an 8-bp sequence, while the enzyme Sau3A I recognizes only a 4-bp sequence.
The length of the recognition sequence indicates how frequently the enzyme will cut,
on average, in a random sequence of DNA. Enzymes with an 8-bp recognition site will
cut every 48
or 65,536 bp; a 4-bp recognition site will occur every 256 bp. Enzymes with
long recognition sequences are therefore also called "rare cutters" and are ideal tools for
mapping eukaryotic genomic DNA.
The majority of enzymes have a palindromic recognition sequence characterized by a
common structural property: a twofold axis of rotational (dyad) symmetry (i.e., a DNA
sequence which is the same when complementary strands are read in opposite direc-
tions). An example is the recognition sequence of the enzyme Swa I:
5'-ATTTAAAT-3'
continued on next page
3
8
Basic Information
Which cleavage sites are recognized by Class II restriction
endonucleases? (continued)
Cleavage sites can be unambiguous or ambiguous. The enzyme BamH I recognizes
unambiguously only the single defined sequence GGATCC. In contrast, Hinf I recog-
nizes the ambiguous 5-bp sequence GANTC, with ,,N" representing any nucleotide.
Other possible ambiguities are sequences that contain one of the pyrimidines or one of
the purines; e.g., in the recognition site of Xho II:
5´ [(A/G) GATC (G/T)] 3´
Xho II is also a good example of an enzyme with another characteristic to be considered
when talking about recognition sites: the recognition site for one enzyme may include
the restriction site for another. Due to its ambiguous sequence, one of the four possible
Xho II sites will also be a recognition site for BamH I (GGATCC), and all four will be
cut by Sau3A I (GATC).
A subgroup of Class II restriction enzymes, called"Class IIS", does not have palindromic
recognition sequences and does not cleave within the recognition site itself. The
enzymes in this subgroup cleave at a certain, but precise distance from their recognition
sequence, e.g., BpuA I:
5´ GAAAGACNN 3´
3´ CTTTCTGNNNNNN 3´
What types of DNA ends are generated by restriction enzyme
cleavage?
Class II restriction enzymes generate three types of DNA ends, all possessing 5´-phos-
phate and 3´-hydroxyl groups:
b Cohesive 5´ ends
For example, ends generated by Hind III:
5´-AAGCTT-3´ A 5´-AGCTT
3´-TTCGAA-5´ TTCGA-5´ A
b Cohesive 3´ ends
For example, ends generated by Kpn I:
5´-GGTACC-3´ GGTAC-3´ C
3´-CCATGG-5´ C 3´-CATGG
b Blunt ends
For example, ends generated by Pvu II:
5´-CAGCTG-3´ CAG-3´ 5´-CTG
3´-GTCGAC-5´ GTC-5´ 3´-GAC
4
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||


continued on next page
9
Basic Information
What types of DNA ends are generated by restriction enzyme
cleavage?(continued)
In contrast to blunt ends, protruding 5´ and 3´ ends are also called "sticky" or "over-
hanging" ends.
Note: In the Roche Applied Science catalog and the "Recognition Sequences of
Restriction Endonucleases" poster, the type of DNA end generated by a restriction
enzyme is indicated by the position of a downward arrow:
b A AGCTT represents an overhanging 5´ end
b GGTAC C represents an overhanging 3´ end
b CAG CTG represents a blunt end
Restriction enzymes that produce the same single-strand fragment ends form enzyme
families. Although the individual enzymes have different recognition sequences, their
overhanging ends are complementary so that their cleavage fragments can be combined
(ligated) with fragments produced by any other member of the same family. An exam-
ple of such a group of enzymes is the GATC family, whose members include BamH I,
Bcl I, Bgl II, Sau3A, and Xho II.
What are isoschizomers?
Restriction enzymes that are isolated from different organisms and recognize identical
sequences are called isoschizomers. Isoschizomeric enzymes that possess different cleav-
age sites in a particular recognition sequence are called neoschizomers (e.g., Sma I
[CCC GGG] and XmaC I [C CCGGG]).
Because isoschizomers are isolated from different bacterial species or strains, they often
have different stabilities or require different optimum reaction conditions. They may
also possess different sensitivities to methylation, which might be helpful when methy-
lation sensitivity can interfere with digestion and cloning steps.
How is the name of a restriction enzyme defined?
The first three letters refer to the organism from which the restriction enzyme was orig-
inally isolated, the fourth letter (if present) refers to the strain, and the Roman numer-
als serve as indices if the same organism contains several different restriction enzymes.
BamH I: Bacillus amyloliquefaciens, strain H, enzyme I
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6
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Basic Information
What is star activity, and when does a restriction enzyme display it?
Under nonstandard conditions, a restriction enzyme cleaves at sites that are similar, but
not identical, to its normal recognition sequence. Such altered cutting is called "star" or
"relaxed" activity.
EcoR I is a well-known example of an enzyme that exhibits star activity. Its normal spe-
cific recognition site is G AATTC; however, under nonstandard conditions the recog-
nition site changes to N AATTN. This sequence will occur with a much higher fre-
quency than the normal one. If the reaction conditions become even more nonstan-
dard, the recognition sequence changes to Pu PuATPyPy, resulting in almost com-
pletely nonspecific digestion of DNA.
Nonstandard conditions include:
b High pH (>8.0) or low ionic strength
b Glycerol concentrations >5% (important, because enzymes are usually delivered as
concentrated stock in 50% glycerol)
b Extremely high concentration of enzyme (>100 U/g of DNA)
b Prolonged incubation time with enzyme
b Presence of organic solvents in the reaction (e.g., phenol, chloroform, ethanol,
DMSO)
b Incorrect cofactor (i.e., Mn2+
, Hg2+
, or Co2+
instead of Mg2+
)
It was previously thought that star activity was only exhibited by certain enzymes; how-
ever, it may be a general characteristic of all restriction enzymes. Information about star
activity is provided in the pack insert of the respective enzyme.
To avoid star activity, always use the optimal buffer system and enzyme amount re-
commended in the pack insert. Make sure that the DNA preparation is free of organic
solvents and contaminating salts.
7
11
Information provided by Roche Applied Science
I am using restriction enzymes for the first time - what do I need to
be aware of?
Mix the restriction enzyme solutions before using them; the 50% glycerol solution is
viscous, so fairly vigorous mixing is required (mix by pipetting - do not vortex). Mixing
is especially important the first time a new vial is used.
Choose which enzyme to use by considering:
b Substrate used
b Site preference
b Methylation
b Ease of use
b Star activity
What experimental setup do you recommend for a basic restriction
enzyme digest?
A standard experiment is set up as indicated under ,,Typical experiment" in each insert
supplied with the respective enzyme e.g.,
Component Final concentration
DNA 1 g
Respective 10 x SuRE/Cut Buffer 2.5 l
Sterile redist. water Up to a total volume of 25 l
Restriction enzyme 1 unit
Incubate at 37°C for one hour.
Which reagents do you recommend for the purification of DNA?
To purify your substrate we recommend to use,
the High Pure PCR Template Preparation Kit
(Cat. No. 11 796 828 001), the DNA
Isolation Kit for Mammalian Blood
(Cat. No. 11 667 327 001) or the DNA Isolation
Kit for Cells and Tissues
(Cat. No. 11 814 770 001) when using genomic DNA.
the Genopure Plasmid Midi or Maxi Kit (Cat. Nos. 03 143 414 001, 03 143 422 001)
when using plasmid DNA.
1
2
3
12
Information provided by Roche Applied Science
What information about quality control is provided by Roche Applied
Science?
Information about quality control and heat inactivation is provided in the pack insert
of the respective enzyme. To see an example, view the pack insert for Sma I at
http://www.roche-applied-science.com/pack-insert/0220566a.pdf
A typical quality control assay consists of the following tests:
b Activity assay
Rigorous definition of unit
activity ensures complete and
specific cutting
Allows economical usage
b Absence of nonspecific endonuclease activities
Ensures reliable and consistent results: no partial digestions, no nonspecific sites
b Absence of exonuclease and phosphatase activity
Ensures intact ends for correct and efficient ligations
continued on next page
4
1g bacteriophage DNA was
incubated for 16 h at 37°C with an
excess (10 to 500 units) of Hind III
from different suppliers. Buffer
conditions were those as recom-
mended by each supplier. The
numbers indicate the amount of
units that were used. The arrows
indicate endonuclease contami-
nation as seen by the appearance
of a smear or undefinded frag-
ments on the gel.
1g bacteriophage DNA was
incubated for 16 h at 37°C with an
excess (10 to 500 units) of Sma I
from different suppliers. Buffer
conditions were those as recom-
mended by each supplier. The
numbers indicate the amounts of
units that were used. The arrows
indicate a downward smear that is
typically caused by exonuclease
or phosphatase contamination.
Endonuclease contamination
Endonuclease contamination
Exonuclease contamination
Endonuclease contamination
Exonuclease contaminationExonuclease contamination
RAS
BA
A
C
C RAS
B
13
Information provided by Roche Applied Science
What information about quality control is provided by Roche Applied
Science? (continued)
b Ligation and recutting assay
Ensures that the correct restriction sequence is preserved
See the restriction enzyme's data label for lot-specific values.
How is one unit of restriction enzyme defined?
The catalytic activity of the restriction endonucleases available from Roche Applied
Science is based on the determination of the minimum amount of enzyme required for
the generation of the enzyme-specific final fragment pattern of a given substrate DNA
(Phage- DNA in most cases).
One unit is the enzyme activity that completely cleaves 1 g of substrate DNA sus-
pended in 50 l of the recommended reaction buffer in 60 minutes at the appropri-
ate temperature under optimal assay conditions as stated for each restriction
endonuclease.
For enzyme-specific parameters, refer to the respective pack insert.
Note: This unit definition is not based on classic enzyme kinetics. A unit defined by this
method measures enzyme activity by an endpoint determination, not via the classical
initial rate term. The molar concentration of the enzyme is in excess. Complete diges-
tion is confirmed on an ethidium bromide-stained gel by visualization of the pattern of
cleaved DNA fragments resolved by electrophoresis.
How stable is a restriction enzyme?
All restriction enzymes are supplied with control data indicated on the label. Roche
Applied Science guarantees 100% activity until the enzyme's respective expiration date.
The majority of restriction enzymes are guaranteed to remain stable for 18 months (a
few for 12 months) from the date of manufacture, if properly stored. Refer to the pack
insert for correct storage conditions, and to the data label on the enzyme's container for
the lot-specific expiration date.
Important: Restriction enzymes are, in general, heat labile and therefore should be kept
at -15 to -20°C. For brief periods of time, the enzymes can be kept on ice or in a small
freezer box.
5
6
After delivery, the shipping package containing the enzyme
remained on the lab bench for two days. Is the enzyme still active?
Our restriction enzymes are always shipped on dry ice. When the enzyme arrives, there
should be dry ice remaining in the shipping package. If the enzyme container still feels
cold to the touch, the enzyme, in most cases, should still be completely active.
Which restriction enzymes have sufficient activity to be used directly
in a PCR mix?
In many cases, our restriction enzymes are fully active in a PCR mix, and therefore are
suitable for direct use in the appropriate restriction analysis. Restriction endonuclease
activity is influenced by the buffer used for PCR as well as the enzyme's ability to cleave
in the presence of primers.
The activity of a restriction enzyme in PCR buffer is indicated in its corresponding pack
insert. An overview table, containing information on the relative activity of our restric-
tion enzymes in a standard PCR mix is provided below.
14
Information provided by Roche Applied Science
7
8
Restriction Recommended Relative Activity
Enzyme SuRE/Cut Buffer (%) in PCR Mix
Acc I A <5
Alu I A 100
Apa I A 100
Asp700 I B 10
Asp718 I B 100
Ava I B 20
Ava II A <5
Avi II H 30
BamH I B 100
BbrP I B 100
Bfr I M 100
Bgl II M 0
Cla I H 100
Dpn I A 100
Dra I M 100
EclX I B 0
Eco47 III H 0
EcoR I H 50
EcoR V B 10
Hae III M 100
Hind II M 100
Hind III B 10
Hinf I H 50
Hpa I A 100
Kpn I L 50
Restriction Recommended Relative Activity
Enzyme SuRE/Cut Buffer (%) in PCR Mix
Ksp I L 0
Mam I H 20
Mlu I H <5
Mvn I M 40
Nae I A 0
Nco I H 50
Not I H 0
Nru I B 75
Pst I H 90
Pvu I H <5
Pvu II M 100
Rsa I L 100
Sac I A 100
Sal I H 0
Sau3A I A 100
Sca I H <5
Sma I A 100
SnaB I M 50
Sph I M <5
Ssp I H 0
Stu I B 30
Sty I H <5
Tag I B 100
Xba I H 60
Xho I H <5
Enzyme activity in an standard PCR Mix
(10 mM Tris/HCl, ph 8.3 at 20°C, 50 mM KCL, 1.5 mM MgCl2
continued on next page
15
Information provided by Roche Applied Science
Which restriction enzymes have sufficient activity to be used directly
in a PCR mix? (continued)
For cloning applications, the direct digestion of the amplified fragment in the PCR mix-
ture without purification is not recommended. Taq DNA Polymerase may still be active
and this will result in polishing of 5´ sticky ends and the addition of an extra dA residue
to the blunt ends of restriction fragments.
Is digestion of hapten-labeled DNA fragments possible?
In principle, a DIG label will interfere with the restriction enzyme's activity if the recog-
nition sequence of the enzyme contains a DIG- or Biotin-dUTP.
The probability of interference also depends on the labeling ratio of DIG-dUTP to
dTTP. For example, a labeling ratio of 1:3 (required to synthesize highly specific
hybridization probes, for example, when using the PCR DIG Probe Synthesis Kit
, Cat.
No. 11 636 090 001) will lead to one DIG moiety in every stretch of 20-25 nucleotides.
The lower the labeling ratio (e.g., 1:10 or 1:20 in the PCR DIG Labeling Mix
,
Cat. No. 11 585 550 001; please note that this kit is not suited for the generation of
hybridization probes), the higher the probability that the recognition sequence will not
be affected.
Is a dU-containing substrate digested by some common restriction
enzymes?
A dU-containing substrate is readily digested by restriction enzymes (e.g., EcoR I und
BamH I), while others show reduced activity (e.g., Hpa I, Hind I, Hind III) on these sub-
strates.
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10
16
Information provided by Roche Applied Science
I want to design a hybridization probe that can be cleaved by a
restriction enzyme. What is the minimum sequence length 5´ and 3´
adjacent to the recognition sequence that is required to ensure suf-
ficient cleavage? Will the enzyme work if the recognition sequence
is close to a biotinylated 5´ end?
Generally, six bases on either side of the recognition sequence are required to ensure
efficient cleavage (use at least eight bases for Nde I and Not I).
When working with biotinylated probes in particular, keep this general rule in mind to
minimize steric hindrance. Another important aspect is the incubation time: in critical
cases an extended incubation time of up to 20 hours can help to improve the cleavage
rate.
The volume of the enzyme in the vial appears to be very low. Could
short-filling or leakage during shipment be the cause?
Some enzymes are supplied in a very low volume; therefore, the vial appears to be
empty. During shipment, the enzyme may be dispersed over the interior surface of the
vial or gathered under the cap. To check whether the volume is correct, perform the fol-
lowing steps while keeping the enzyme cooled to 4°C:
b Carefully check the exterior of the enzyme vial, noting any glycerol leakage.
b Prepare a water blank containing the enzyme's expected volume to use as a coun-
terbalance.
b Briefly spin the enzyme and blank in a microcentrifuge.
b With both vials on ice, estimate the volume of the enzyme by comparison to that
of the blank.
How can I calculate the optimum number of enzyme units for my
restriction digest?
Reference DNA is used in the specific unit assay for each restriction enzyme. This ref-
erence DNA is identified in the pack insert, in addition to the number of cleavage sites
for the respective enzyme in this reference DNA.
As an example, for EcoR I, one unit of EcoR I is defined as the amount that completely
cleaves 1 g DNA in 1 hour at 37°C. Since DNA has a size of 48,502 bp and six cleav-
age sites for EcoR I, one unit of EcoR I, is, in general, sufficient to cleave DNA with an
average cleavage-site frequency of 1/8084 bp.
continued on next page
11
12
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17
Information provided by Roche Applied Science
How can I calculate the optimum number of enzyme units for my
restriction digest? (continued)
If you want to cleave 2.5 g of a 4363-bp plasmid carrying two EcoR I sites, this corre-
sponds to a site frequency of 1/2181 bp. Thus, for complete digestion of 1 g of your
plasmid in 1 hour, you would need approximately fourfold more enzyme units than for
digesting DNA. Multiplied by the DNA amount of 2.5 g in your assay, this results in
a total of 10 units you should apply in your assay.
Keep in mind also, that the nature of the substrate strongly influences the activity of
restriction enzymes. For plasmid DNA, especially, you must consider the topology of
DNA: supercoiled plasmids need more unit activity (up to fivefold; even 15-fold for Sfi
I) for complete cleavage compared to linearized DNA.
What do you recommend for dilution of restriction enzymes, and how
stable are diluted restriction enzymes?
In order to obtain the correct concentration, enzymes are often diluted prior to addi-
tion to the reaction mixture. The following dilution buffer is recommended: 20 mM
potassium phosphate buffer (pH 7.4), 200 mM KCl, 1 mM EDTA, 7 mM -mercapto-
ethanol, 10% glycerol and 0.2mg/ml BSA. After dilution with this buffer, the enzyme
should be used within a day! Longtime storage is not recommended. Do not freeze the
diluted enzyme.
Alternatively, restriction enzymes may also be diluted in the storage buffer that is indi-
cated in the pack insert for each enzyme, and left for several weeks to months at -20°C.
Do not dilute to a concentration of less than 1 U/l. Note that for some enzymes, espe-
cially EcoR I, drastic loss of activity within two days after dilution in storage buffer (5
U/l) has been reported. Therefore, stability of enzymes after dilution in storage buffer
cannot be guaranteed.
Some laboratory manuals recommend dilution of enzymes with 1x reaction buffer
before addition to the DNA. Such dilution might partially or completely inactivate the
enzyme, especially if the reaction buffer has low ionic strength and contains no stabi-
lizing agents such as BSA. Do not dilute restriction enzymes in reaction buffer.
Always add the restriction endonuclease last to the reaction mixture. Appropriate mix-
ing of the enzyme is recommended before adding it to the reaction mixture; however,
do not vortex dilutions or the final reaction mixture. Mix by gentle pipetting and avoid
generation of air bubbles.
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18
Standard Digest
What are the conditions for a successful restriction enzyme digest?
Make sure your DNA preparation is pure and free of contaminants such as phenol,
chloroform, ethanol, detergents, EDTA, and salts because these can partially or com-
pletely inhibit the activity of the restriction endonuclease. Such compounds should be
completely removed by ethanol precipitation followed by drying, before the DNA is
added to the restriction digest reaction.
b Optimal reaction conditions for each enzyme are indicated in the pack insert. They
can also be found in the Roche Applied Science Lab FAQs guide or the Laminated
Buffer Chart.
b Always use the recommended reaction buffer supplied with the enzyme. Low
enzyme activity may sometimes be due to deterioration of the reaction buffer; in
such cases, using fresh buffer will solve the problem.
b When working with a restriction enzyme, always keep it on ice and add it last to the
digestion reaction mixture, using a fresh pipette tip.
How can the substrate influence the restriction digest?
In addition to the incubation conditions, the nature of the substrate strongly influences
the activity of restriction enzymes. Important general parameters of the substrate are:
b Base distribution in natural DNA
b Site density per g DNA
b Tertiary DNA structure
b Base composition of the flanking sequences
b Position of the cleavage sites with respect to each other
b Methylation status of the substrate
Flanking sequences: Restriction endonucleases differ in their ability to cleave at recog-
nition sites close to the end of a DNA fragment. Cleavage close to the end of a fragment
is important when two restriction sites are close together in the multiple cloning site of
a plasmid or when cleaving near the ends of PCR products or oligonucleotides. Many
restriction enzymes can cleave near a DNA end having one base pair remaining in addi-
tion to a 1 to 4 single-base overhang produced by an initial cleavage; others require at
least 3 base pairs in addition to an overhang. When designing PCR primers containing
restriction sites, adding eight random bases 5´ of the restriction site is recommended for
complete digestion of the restriction sites. However, in these cases, higher quantities of
enzyme and longer reaction times are also necessary. Normal reaction rates are achieved
only with increasing length of the flanking sequences.The base composition within the
flanking sequences is also important for the cleavage rates at the individual cleavage
sites.
Plasmids: Supercoiled plasmids often require more restriction endonuclease to achieve
complete digestion than linear DNA.
1
2
Double Digest
Digestion with multiple enzymes: would you recommend performing
this simultaneously or sequentially?
Digesting DNA with two restriction enzymes is a common task, and often the two
enzymes have different buffer requirements. Here are some recommendations for per-
forming double digests:
b Digest with both enzymes in the same buffer
In many cases, even if one given buffer is not optimal for an enzyme, cleavage rates
are still quite good. Roche Applied Science provides a double-digest table (see
below) which lists the best single buffer for performing specific double digests. If the
reaction produces extra fragments, possibly caused by star activity, reduce the reac-
tion time or the amount of enzyme. If the reaction is incomplete, test each enzyme
individually to determine its ability to linearize the plasmid. A lack of cutting may
indicate an inactive enzyme, absence of the expected site, or inhibitors in the tem-
plate preparation. Test the enzyme on a second target as a control. If both enzymes
are active and the restriction sites are within several bases of each other, there may
be a problem cutting close to the end of the fragment.
b Cut with one enzyme, then alter the buffer composition and cut with the
second enzyme
Always perform the first digest with the enzyme requiring the lower salt buffer. Then
adjust the buffer composition for the needs of the second enzyme and add the
required amount of salt.
b Change buffer between digestions with two enzymes
Perform one digestion, recover the DNA (by ethanol precipitation), then resuspend
it in the buffer appropriate for the second enzyme.
b Prepare a sequential digest (with or without altering buffer composition) if
using restriction enzymes requiring different reaction temperatures.
Double Digestion Table
Enzyme Asp718 I BamH I Bgl II Bln I Bst1107 I BstX I
2
Cla I Dpn I Dra II Eco47 III EcoR I EcoR V Hind III Kpn I
1
Mlu I Nae I Nco I Nde I Nhe I Not I Nsp I Pst I Pvu I Pvu II Sac I Sal I Sfi I
3
Sma I
4
Spe I Sph I Stu I Xba I
100% Activity
in Sure/
Cut Buffers B ABM ABMH H H BH ABMH A AL H ABH B BM L H AL H H ALM H M H H M AL H M A MH M ABL AH
BamH I A B M B
Bgl II A B M H B M
Bln I H H B H
Bst1107 I H H B H H
BstX I
2
B H B B H H H
Cla I A B M H B B H H H H
Dpn I A A A A H H H A
Dra II A L A A A B B B A A
Eco47 III H H B H H H H H H SD
EcoR I A B H B A H H H H H A A H
EcoR V B B B B H H B B B B H B
Hind III B M B B B H H B B B A H B B
Kpn I
1
L A L A SD SD SD A A L SD A SD A
Mlu I H H H H H H H H H SD H H H H SD
Nae I A L A A A A A SD A A L SD A SD A L SD
Nco I H H B H H H H H H L H H H H L H L
Nde I H B B H H H H H H B H H H B SD H A H
Nhe I A L M A M A A M B M A A SD A M M L SD L L M
Not I H H B H H H H H H SD H H H H SD H SD H H SD
Nsp I M B M M B B B M M L B M B M L SD L M M M B
Pst I H H H H H H H H H A H H H H SD H A H H M H M
Pvu I H B B H H H H H H A H H B B L H A H H A H M H
Pvu II M M M M H H H M M M SD M M M M H A M M M H M M M
Sac I A L A A A A A SD A A A SD A A A L SD L A M L SD L A A M
Sal I H H H H H H H H H SD H H H H SD H SD H H SD H SD H H H SD
Sfi I
3
M M M M M H B M M L SD M M M L H L M M M M M M M M L H
Sma I
4
A A A A A A SD A A A SD A A A A SD A A A A SD A A A A A SD A
Spe I M H B M H H H H H H L H H H M L H A H H M H M H H M A H M A
Sph I M B M M H H H M M A H M B M A H A H H M H M H H M M H M A M
Stu I A B L B A A H H B B A L H A B B L H L H B L H M H B M L H M A L M
Xba I A H B A H H H H H A A H H B B A H A H H A H M H H M A H M A H M A
Xho I H B B H H H H H H B H H B M L H A H H M H M H H H A H M A H H B H
Legend: SD Sequential digest is recommended.
One enzyme is less than 50% active in specified buffer.
1
Enzyme requires addition of BSA.
2
Enzyme requires incubation at 45°C.
3
Enzyme requires incubation at 50°C.
4
Enzyme requires incubation at 25°C.
Please remember that star activity may be activated by high glycerol concentration, significant overdigestion, and long incubation times (>16 h).
20
Genomic Digest
After restriction enzyme digestion of genomic DNA to be subse-
quently used in a Southern blot, how can you check if digestion is
complete?
A complete digestion is characterized by a stable pattern, to be distinguished from an
incomplete or degraded pattern. The digestion is complete when a similar pattern of
DNA fragmentation appears in consecutive samples of decreasing enzyme concentra-
tion within the serial digest. If samples with high enzyme concentration show smears
that contain fragments smaller than those seen in samples containing less enzyme, then
most probably degradation is occurring. If the sample containing the most enzyme is
the only sample demonstrating a complete digest, then the subsequent samples (con-
taining less enzyme) will demonstrate progressively larger fragments. A uniformly
banded pattern will not occur in serial samples unless the samples are all completely cut
or completely uncut.
For a detailed protocol how to perform a Southern blot in combination with DIG
labeled probes please refer to our website:
http://www.roche-applied-science.com/DIG
or the DIG Application Manual for Filter Hybridization.
1
21
DNA Methylation
What is meant by DNA methylation, and how can it affect a restric-
tion enzyme digest?
DNA methylation is the covalent modification of DNA by the transfer of a methyl
group from S-adenosylmethionine to one of a few possible sites on cytosine or ade-
nine.
DNA methylation is involved in several biological processes, including restriction
and modification, mismatch error correction (a DNA repair process), and the con-
trol of eukaryotic gene expression.
In prokaryotes, both adenine and cytosine methylation occurs, whereby N6
-methyl-
adenine is the major methylated base and to a lesser extent N4
-methylcytosine.
Methylation in bacteria serves two functions: either to protect special sites against
cleavage by restriction endonucleases or to correct mismatch error (as methylation
of A residues in the sequence 5´-GATC-3´ in E. coli). Furthermore, it plays a role in
controlling initiation of DNA replication.
In eukaryotes, methylation occurs at the 5´ position of the pyrimidin ring of
cytosines.
In animals, methylation is found primarily in C residues that are immediately 5´ to
G residues (that is, in the sequence CpG).When such a C is methylated, so is the cor-
responding C in the complementary strand. In plant DNA, in addition to CpG, the
methylated sequence can also be CpNpGp with N being any base. In comparison to
animals, plants have a much higher proportion of methylated cytosine (30% versus
2 - 5%).
Depending on the particular strain, E. coli expresses four different methylases that
are able to methylate adenines or cytosines which ­ when located in a cleavage site
­ may inhibit cleavage by a restriction enzyme:
b The most widely distributed methylase systems are the M-Eco dam I methylase
(encoded by the dam gene) and the M-Eco dcm I methylase (encoded by the
dcm gene). The dam methylase recognizes the sequence GATC and methylates
the N6
position of adenine. The dcm methylase recognizes the sequence
CC(A/T)GG and methylates the internal cytosine at the C5
position. E. coli may
harbor both methylases at the same time.
b The EcoB and EcoK methylases are encoded by the ecoB/ecoK gene located on the
same locus. Therefore, E. coli can only contain EcoB or EcoK, not both at the
same time. Both methylases have rather long and therefore rare methylation sites
(Am6
ACGTGC and GCm6
ACGTT), and do not play a major role in affecting
restriction enzyme cleavage.
continued on next page
1
22
DNA Methylation
What is meant by DNA methylation, and how can it affect a restric-
tion enzyme digest? (continued)
Thus, plasmid DNA from normal strains may be cleaved partially or not at all by
restriction endonucleases that are sensitive to methylation. This can be avoided by
preparing plasmid DNA from strains that lack these methylases.
Methylation problems can also arise when working with mammalian or plant DNA.
Methylation patterns in eukaryotic genomic DNA can be investigated by using the
different methylation sensitivities of isoschizomers (for example, Mbo I and Sau3A I
are isoschizomers that recognize and cleave the sequence GATC, identical to the
recognition sequence of dam methylase; however, while digestion of Gm
ATC by Mbo I
is completely inhibited, digestion by Sau3A I is unaffected by methylation).
Note:
b A list of the most important cloning strains and information about methylation
sensitivity is always provided in the pack insert of the respective enzyme.
b A sub-group of Class II restriction enzymes (Type IIM) requires a methylated recog-
nition site for cleavage (an example is Dpn I).
I would like to know whether the restriction enzyme I want to use is
sensitive to the methylation pattern of my DNA. Can you explain how
to use the symbols of your "Recognition Sequences of Restriction
Enzymes" poster in this respect?
Methylation will interfere with your restriction digestion when cutting DNA cloned in
dam+
or dcm+
E. coli strains, or when cutting genomic DNA from certain eukaryotic
organisms.
The +
symbol above an A or C in the recognition sequence indicates that cleavage by the
corresponding enzyme is inhibited by N6
-methyladenine or 5-methylcytosine. The
methylation of either A or C in the recognition sequence may occur in E. coli strains fea-
turing dam or dcm methylation. Note that the recognition sequence of the restriction
enzyme doesn't have to include the entire methylation site to be blocked. Overlapping
of the methylation site with the recognition sequence will also cause problems. Xba I,
for example, has the recognition sequence 5´TCTAGA3´ which lacks the GATC dam
methylase target site. Nevertheless, if the preceding 5´ two bases are GA, giving GATC-
TAGA, or the following 3´ bases are TC, giving TCTAGATC, then the dam methylase will
block Xba I from cutting.
The m
symbol above the A or C in a recognition sequence indicates that an N6
-methyl-
adenine or 5-methylcytosine is required for cleavage. In this case, the enzyme you are
intending to use requires a methylated A or C in its sequence to cut efficiently.
continued on next page
2
I would like to know whether the restriction enzyme I want to use is
sensitive to the methylation pattern of my DNA. Can you explain how
to use the symbols of your "Recognition Sequences of Restriction
Enzymes" poster in this respect?(continued)
The ° symbol above an A or C indicates that cleavage is not influenced by N6
-methyl-
adenine or 5-methylcytosine. If A or C are not marked by a symbol, the influence of
methylation on restriction enzyme activity is still unknown or ambiguous.
A dotted line under a sequence (e.g., Bcl I TGATCA) indicates the complete or partial
recognition sequence of the E. coli dam-gene encoded methylase M-Eco dam I.
A dotted line above a sequence (e.g., Sex AI ACC(A/T)GGT) indicates the complete or
partial recognition sequence of the E. coli dcm-gene encoded M-Eco dcm I methylase.
If the M-Eco dam or M-Eco dcm methylation sequence is only partial, the flanking
nucleotides must complete the GATC or CC(A)GG complement in order for cleavage
inhibition to occur.
Note that the presence of a methylase site within a cleavage site does not mean that the
corresponding enzyme is in any case inhibited by methylation: while BamH I is not
inhibited by GGAm6
TCC, Bcl I is inhibited by TGAm6
TCA.
23
DNA Methylation
24
Troubleshooting
After analyzing the restriction enzyme digest by gel electrophoresis,
I observe no cleavage at all. What could be the cause for this?
b Check the amount and nature of your DNA. See the FAQs "How can I calculate the
optimum number of enzyme units for my restriction digest?" and "How can the
substrate influence the restriction digest?" for details.
b Check the purity of your template. Residual inhibiting compounds like EDTA, phe-
nol, chloroform, ethanol, CsCl, NaCl, or metal ions in the substrate DNA solution
could interfere with the reaction (see the FAQ concerning star activity).
To purify your substrate, use
· the High Pure PCR Template Preparation Kit
, the DNA Isolation Kit for
Mammalian Blood
, or the DNA Isolation Kit for Cells and Tissues
when
using genomic DNA
· the Genopure Plasmid Midi or Maxi Isolation Kit when using plasmid DNA.
b Is your DNA methylated and your enzyme sensitive to methylation? Digest your
DNA with an isoschizomer that is insensitive to methylation. If you are working
with plasmid DNA, use a host strain which lacks dam and dcm methylases.
b Check the temperature chosen for the digest. Some restriction enzymes require
special incubation temperatures (e.g., Sfi I (50°C) or Sma I (25°C). The Laminated
Buffer Chart lists all enzymes with incubation temperatures other than 37°C.
b If the enzyme cleaves a particular substrate poorly, the enzyme's activity can be
checked by using DNA along with the test DNA mixed with DNA. If the enzyme's
activity on DNA alone corresponds to that indicated in the pack insert, and the
DNA mixed with the DNA of interest is digested poorly, the DNA should be re-
purified.
b Check if the enzyme's expiration date has been exceeded.
b Check if the enzyme's storage conditions were optimal. Always keep restriction
enzymes on ice, even during setup of the reaction.
b Check the dilution and addition of enzyme. Some restriction enzymes are very sen-
sitive to the concentration of glycerol in the reaction mixture. Since our restriction
enzymes are supplied in 50% glycerol, the enzyme should comprise not more than
1/10 of the final reaction volume (i.e., no more than 5% glycerol). Always add the
restriction enzyme last to the reaction mixture. Mix gently. Ensure thorough mixing
of the reaction, but do not vortex.
1
25
Troubleshooting
What could be the reason for partial cleavage?
In this case, check the following:
b Is the substrate DNA pure enough? The efficiency of the restriction endonuclease
reaction is very dependent upon the purity of the substrate DNA. Contaminants
found in some DNA preparations (e.g., protein, phenol, chloroform, ethanol, EDTA,
SDS, CsCl, high salt concentration) may inhibit restriction endonuclease activity.
b After addition of restriction enzyme to the reaction mixture, did you thoroughly
mix to ensure even distribution?
b Is the concentration of substrate DNA too high for the applied number of enzyme
units?
b Is the substrate DNA supercoiled? In this case you could add more enzyme (up to
20 fold) to the reaction mix, as supercoiled DNA requires more units of enzyme for
complete digestion. This also applies to some viral DNAs.
b Some special enzymes show site preferences on different substrates or are influenced
by sequences flanking the cleavage site. Several cleavage sites on these DNAs are
cleaved at extremely slow rates and complete digestion is obtained only with excess
of enzyme units (examples are Nae I, Nar I, Sac II, Xma III) For more information,
please refer to the respective pack insert.
It seems that the enzyme I am using has a lower enzyme activity than
stated.
b Make sure that you followed exactly the instructions in the pack insert (e.g., reaction
temperature, buffer system, and correct storage and handling of enzyme).
b Is the substrate DNA supercoiled? In this case you could add more enzyme to the
reaction mix, as supercoiled DNA requires more units of enzyme for complete
digestion.
b If the enzyme was diluted prior to use, check the dilution.
Note: Diluted enzymes which are not diluted in storage buffer should be used with-
in one day.
2
3
26
Troubleshooting
Additional DNA bands not typical for the expected fragment pattern
appeared on my gel. What can I do?
b Star activity could be the reason for your problem. A restriction enzyme shows star
activity under non-optimal conditions such as low ionic strength, high pH of the
reaction buffer, excess concentration of enzyme, excess concentration of glycerol,
manganese, or divalent cations other than magnesium. Under these conditions,
restriction enzymes begin to cleave the substrate in other sites in addition to the
normally recognized sequences.You should observe additional bands lower than the
expected bands and no additional bands higher than the largest fragment. Addition
of more enzyme and a prolonged incubation time will lead to an increase in addi-
tional bands and decrease of the typical banding pattern, if star activity is the cause
of your problem.
Note: To avoid star activity, add less enzyme, perform the reaction in the recom-
mended buffer, and avoid a too-high concentration (>5%) of glycerol in the reac-
tion mix. See the FAQ on star activity for details.
b If low-intensity bands are present above the expected bands on the gel and there are
no bands below the smallest band, then your RE digest is incomplete. Increase the
incubation time and the amount of enzyme, and the bands will disappear.
b If following these suggestions do not help you to achieve the correct banding pat-
tern, it could be that
your enzyme is contaminated with another enzyme, which can occur due to
improper handling,
or the substrate DNA is contaminated with DNases, which is often the case for
"miniprep" plasmid preparations.
After digestion there is only a smear on my gel - what went wrong?
This indicates a probable nuclease contamination from the bacterial host or the
reagents used. Work under sterile conditions, wear gloves, and do not re-use tips.
How can I set up a control reaction?
Control reactions are an essential part of good laboratory practice.
For your restriction enzyme digest, prepare one vial containing sample DNA reaction
buffer and no restriction enzyme. DNA degradation in your control vial indicates that
you might have a nuclease contamination in your reaction buffer or the DNA prepara-
tion.
4
5
6
27
Troubleshooting
The ligation efficiency is poor. What could be the reason?
There could be several reasons for this, such as:
b Degradation of ligation buffer components such as ATP and DTT. Repeat the liga-
tion with fresh buffer.
b The restriction enzyme is still active in the ligation mixture. Heat inactivate the RE
reaction mixture after digestion at 65°C; treat with phenol/chloroform, and/or pre-
cipitate with ethanol before ligation, depending on the possibility of inactivating the
restriction enzyme by heating.
b Check if the DNA preparation, the ligation reaction, or the restriction enzyme reac-
tion is contaminated with nuclease.
b Check the ligase concentration. Ligation of blunt-ended fragments need more ligase
activity (0.1-0.5 units/l).
b Has the vector ligated to itself? If the vector has compatible ends: Were the 5´ phos-
phate groups removed by dephosphorylation and was the success of dephosphory-
lation checked?
b Another reason for a failed ligation could be that the molar ratio of vector and insert
is not sufficient. For suggestions, see the pack insert of our Rapid DNA Ligation Kit.
b Check if the competent cells used are really functional. Perform a control transfor-
mation.
7
28
References
Williams, R.J.
Mol. Biotechnol. 23: 225-243 (2003).
Restriction endonucleases: Classification, properties, and applications.
Jeltsch, A.
Chembiochem 3: 274-293 (2002).
Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA
methyltransferases.
Perona, J.J.
Methods 28: 353-364 (2002).
Type II restriction endonucleases.
Pingoud, A., Jeltsch, A.
Nucleic Acids Res. 29: 3705-3727 (2001).
Structure and function of type II restriction endonucleases.
Raleigh, E.A., Brooks, J.E.
(1998) In Bacterial Genomes (De Bruijn, F.J., Lupski, J.R., Weinstock, G.M. Ed.). pp
78-92. Chapman & Hall, New York.
Restriction modification systems: where they are and what they do.
Smith, D.R.
(1996) In Methods Mol. Biol. Vol 58.
(A. Harwood Ed.). pp 11-15. Humana Press, Inc., Totowa, NJ.
Restriction endonuclease digestion of DNA.
Palmer, B.R., Marinus, M.G.
Gene 143: 1-12 (1994).
The dam and dcm strains of Escherichia coli - a review.
Bhagwat, A.S.
Methods Enzymol. 216: 199-224 (1992).
Restriction enzymes: Properties and Use.
Bird, A.
Cell 70: 5-8 (1992).
The essentials of DNA methylation.
Roberts, R.J., Macelis, D.
Nucleic Acids Res. 19: 2077-2109 (1991).
Restriction enzymes and their isoschizomers.
Kessler, C., Manta, V.
Gene 92: 1-248 (1990).
Specificity of restriction endonucleases and DNA modification methyltransferases -
a review (Edition 3).
Gerstein, AS.
Molecular Biology Problem Solver: A Laboratory Guide. Chapter 9: Restriction
Endonucleases (2001) by Wiley-Liss, Inc.
ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic).
Ausubel, FM., Brent, R., Kingston, RE., Moore DD, J. G. Seidmann JG, Smith JA.,
Struhl K. (Eds)
Current Protocols in Molecular Biology (1998)
by Whiley & Sons; Four Volumes 0-471-50338-X-Looseleaf or 0-471-30661-4-CD-Rom.
29
Ordering Information
Aat I see isoschizomer Stu I
Aat II GACGT C 1­5 10 775 207 001 250
Acc I GT (A,C)(T,G)AC 5 10 728 420 001 100
5 10 728 438 001 500
Acc III see isoschizomer Mro I
Acs I (A,G) AATT(T,C) 10 11 526 456 001 200
Acy I (Aha II) G(A,G) CG(C,T)C 5 11 081 314 001 200
Afl I see isoschizomer Ava II
Afl II see isoschizomer Bfr I
Afl III A C(A,G)(T,C)GT 5 11 209 183 001 100
Age I see isoschizomer PinA I
Aha II see isoschizomer Acy I
Aha III see isoschizomer Dra I
Alu I AG CT 10 10 239 275 001 500
10 10 656 267 001 2000
Alw44 I (Sno I) G TGCAC 10 11 450 506 001 1000
Aos I see isoschizomer Avi II
Apa I GGGCC C 10 10 899 208 001 5000
40 10 703 745 001 5000
40 10 703 753 001 20000
ApaL I see isoschizomer Alw44 I
Apo I see isoschizomer Acs I
Apy I see isoschizomer EcoR II, Mva I
Asp I (Tth111 I) GACN NNGTC 10 11 131 354 001 400
Asp700 (Xmn I) GAANN NNTTC 10 10 835 277 001 500
10 10 835 285 001 2500
Asp718 G GTACC 10 10 814 245 001 1000
10 10 814 253 001 5000
40 11 175 050 001 5000
AspE I GACNNN NNGTC 10 11 428 179 001 200
Asu II see isoschizomer Sfu I
Ava I C (T,C)CG(A,G)G 5 10 740 721 001 200
5 10 740 730 001 1000
Ava II G G(A,T)CC 5 10 740 756 001 500
Avi II (Aos I) TGC GCA 10 11 481 436 001 200
Avr II see isoschizomer Bln I
Bal I see isoschizomer MluN I
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
30
Ordering Information
BamH I G GATCC 10 10 220 612 001 1000
10 10 567 604 001 2500
10 10 656 275 001 10000
40 10 798 975 001 10000
40 11 274 031 001 50000
Ban II G(A,G)GC(T,C) C 10 10 775 240 001 500
BbrP I (PmaC I) CAC GTG 10 11 168 860 001 500
Bbs I see isoschizomer BpuA I
Bcl I T GATCA 10 10 693 952 001 500
10 10 693 979 001 2500
40 11 097 059 001 2500
Bfr I (Afl II) C TTAAG 10 11 198 939 001 500
Bgl I GCC(N)4 NGGC 10 10 404 101 001 1000
10 10 621 641 001 5000
40 11 047 604 001 5000
Bgl II A GATCT 10 10 348 767 001 500
10 10 567 639 001 2000
40 10 899 224 001 2000
40 11 175 068 001 10000
Bln I (Avr II) C CTAGG 10 11 558 161 001 200
10 11 558 170 001 1000
BpuA I GAAGAC(N)2/6 10 11 497 944 001 200
BseA I T CCGGA 10 11 417 169 001 200
BseP I see isoschizomer BssH II
BsiW I C GTACG 10 11 388 959 001 300
BsiY I CCNNNNN NNGG 10 11 388 916 001 200
Bsm I GAATGCN N 10 11 292 307 001 200
Bsp1407 I see isoschizomer SspB I
BspH I see isoschizomer Rca I
BspLU11 I A CATGT 10 11 693 743 001 200
BssH II G CGCGC 10 11 168 851 001 200
BssG I see isoschizomer BstX I
Bst1107 I GTA TAC 10 11 378 953 001 200
BstB I see isoschizomer Sfu I
BstE II G GTNACC 10 10 404 233 001 500
BstN I see isoschizomer Mva I, EcoR II
BstX I CCA(N)5 NTGG 10 11 117 777 001 250
10 11 117 785 001 1250
Cel II (Esp I) GC TNAGC 10 11 449 397 001 200
Cfo I (Hha I) GCG C 10 10 688 541 001 1000
10 10 688 550 001 5000
Cfr I see isoschizomer Eae I
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
31
Ordering Information
Cla I AT CGAT 10 10 404 217 001 500
10 10 656 291 001 2500
40 11 092 758 001 2500
Dde I C TNAG 10 10 835 293 001 200
10 10 835 307 001 1000
Dpn I GA TC 10 10 742 970 001 200
10 10 742 988 001 1000
Dra I TTT AAA 10 10 779 695 001 1000
10 10 827 754 001 5000
40 11 175 076 001 10000
Dra II (A,G)G GNCC(T,C) 1­3 10 843 512 001 250
Dra III CACNNN GTG 1­5 10 843 539 001 100
1­5 10 843 547 001 500
Eae I (Cfr I) (T,C) GGCC(A,G) 10 11 062 557 001 200
Eag I see isoschizomer EclX I
Eam1105 I see isoschizomer AspE I
EclX I C GGCCG 10 11 131 389 001 200
(Xma III) 10 11 131 397 001 1000
Eco47 III AGC GCT 5 11 167 103 001 100
EcoR I G AATTC 10 10 703 737 001 5000
10 11 175 084 001 10000
40 10 200 310 001 10000
40 10 606 189 001 50000
EcoR II CC(A,T)GG 10 11 427 881 001 200
EcoR V GAT ATC 10 10 667 145 001 2000
10 10 667 153 001 10000
40 11 040 197 001 10000
Esp I see isoschizomer Cel II
FnuD II see isoschizomer Mvn I
Fnu 4H I see isoschizomer Ita I
Fok I GGATG(N)9 1­5 11 004 816 001 100
CCTAC(N)13
Fsp I see isoschizomer Avi II
Hae II (A,G)GCGC (T,C) 5 10 693 910 001 100
Hae III GG CC 10 10 693 936 001 1000
10 10 693 944 001 5000
Hha I see isoschizomer Cfo I
Hinc II see isoschizomer Hind II
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
32
Ordering Information
Hind II GT(T,C) (A,G)AC 3­10 10 567 655 001 500
3­10 10 656 305 001 2500
Hind III A AGCTT 10 10 656 313 001 5000
10 10 656 321 001 10000
40 10 798 983 001 10000
40 11 274 040 001 50000
Hinf I G ANTC 10 10 779 652 001 1000
10 10 779 679 001 5000
40 11 097 067 001 5000
40 11 274 082 001 20000
Hpa I GTT AAC 3­10 10 380 385 001 100
3­10 10 567 647 001 500
Hpa II C CGG 10 10 239 291 001 1000
10 10 656 330 001 5000
40 11 207 598 001 5000
Ita I GC NGC 10 11 497 979 001 200
Kpn I GGTAC C 10 10 899 186 001 5000
40 10 742 945 001 2000
40 10 742 953 001 10000
Ksp I CCGC GG 10 11 117 807 001 1000
(Sac II)
Ksp632 I CTCTTC(N)1 10 11 081 276 001 200
GAGAAG(N)4
Mae I C TAG 1­5 10 822 213 001 50
1­5 10 822 221 001 250
Mae II A CGT 1­5 10 862 495 001 50
Mae III GTNAC 1­5 10 822 230 001 50
1­5 10 822 248 001 250
Mam I GATNN NNATC 10 11 131 281 001 200
Mbo I see isoschizomer Nde II
Meganuclease I-Sce I 8­12 11 362 399 001 1000
Mfe I see isoschizomer Mun I
Mlu I A CGCGT 10 10 909 700 001 500
10 10 909 718 001 2500
40 11 207 601 001 2500
MluNI (Bal I) TGG CCA 10 11 526 430 001 200
Mro I (Acc III) T CCGGA 1­5 11 102 982 001 100
Msc I see isoschizomer MluN I
Mse I see isoschizomer Tru9 I
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
33
Ordering Information
Msp I C CGG 10 10 633 518 001 1000
10 10 633 526 001 5000
40 11 047 647 001 5000
Mst I see isoschizomer Avi II
Mun I (Mfe I) C AATTG 10 11 441 337 001 200
Mva I (BstN I) CC (A,T)GG 10 11 288 067 001 1000
10 11 288 075 001 5000
Mvn I (FnuD II) CG CG 10 11 062 573 001 200
Nae I GCC GGC 10 10 786 314 001 250
10 10 786 322 001 1000
Nar I GG CGCC 10 11 103 016 001 200
10 11 103 024 001 1000
Nco I C CATGG 10 10 835 315 001 200
10 10 835 323 001 1000
40 11 047 698 001 1000
Nde I CA TATG 10 11 040 219 001 200
10 11 040 227 001 1000
Nde II (Mbo I) GATC 5 11 040 235 001 200
5 11 040 243 001 1000
Nhe I G CTAGC 10 10 885 843 001 200
10 10 885 851 001 1000
40 10 885 860 001 1500
Not I GC GGCCGC 10 11 014 706 001 200
10 11 014 714 001 1000
40 11 037 668 001 1000
Nru I TCG CGA 10 10 776 769 001 200
10 10 776 777 001 1000
Nsi I ATGCA T 10 10 909 831 001 200
10 10 909 840 001 1000
40 11 207 628 001 1000
Nsp I (A,G)CATG (T,C) 10 11 131 419 001 200
Nsp II see isoschizomer Bmy I
Nsp V see isoschizomer Sfu I
PinA I (Age I) A CCGGT 10 11 464 841 001 200
10 11 464 850 001 1000
PmaC I see isoschizomer BbrP I
Pml I see isoschizomer BbrP I
Psp1406 I AA CGTT 5 11 533 860 001 200
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
34
Ordering Information
Pst I CTGCA G 10 10 621 625 001 3000
10 10 621 633 001 10000
40 10 798 991 001 10000
40 11 274 066 001 50000
Pvu I CGAT CG 5 10 650 137 001 100
5 10 650 129 001 500
Pvu II CAG CTG 10 10 642 690 001 1000
10 10 642 703 001 5000
40 10 899 216 001 5000
Rca I (BspH I) T CATGA 5 11 467 123 001 200
Rsa I GT AC 10 11 729 124 001 1000
10 11 729 132 001 5000
40 11 047 671 001 5000
Rsr II CG G(A,T)CCG 10 11 292 587 001 200
10 11 292 595 001 1000
Sac I (Sst I) GAGCT C 10 10 669 792 001 1000
10 10 669 806 001 5000
40 11 047 655 001 5000
Sac II see isoschizomer Ksp I
Sal I G TCGAC 10 10 348 783 001 500
10 10 567 663 001 2500
40 11 047 612 001 2500
Sau3A I GATC 1­5 10 709 743 001 100
1­5 10 709 751 001 500
Sau96 I G GNCC 10 10 651 303 001 300
Sca I AGT ACT 10 10 775 258 001 500
10 10 775 266 001 2500
40 11 207 636 001 5000
ScrF I (Dsa V) CC NGG 10 11 081 292 001 500
SexA I A CC(A,T)GGT 10 11 497 995 001 200
Sfi I GGCC(N)4 NGGCC 10 11 288 016 001 250
10 11 288 024 001 1250
40 11 288 032 001 1250
40 11 288 059 001 5000
Sfu I (Asu II) TT CGAA 10 11 243 497 001 2000
SgrA I C(A,G) CCGG(T,C)G 10 11 277 014 001 200
Sma I CCC GGG 10 10 220 566 001 1000
10 10 656 348 001 5000
40 11 047 639 001 5000
SnaB I TAC GTA 10 10 997 480 001 200
Sno I see isoschizomer Alw44 I
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
35
Ordering Information
Spe I A CTAGT 10 11 008 943 001 200
10 11 008 951 001 1000
40 11 207 644 001 1000
Sph I GCATG C 10 11 026 950 001 200
10 10 606 120 001 500
10 11 026 534 001 2500
40 11 026 542 001 2500
Ssp I AAT ATT 10 10 972 967 001 200
10 10 972 975 001 1000
40 11 207 652 001 1000
SspB I T GTACA 10 11 497 901 001 200
Sst I see isoschizomer Sac I
Sst II see isoschizomer Ksp I
Stu I AGG CCT 10 10 753 351 001 500
10 10 753 360 001 2500
40 11 047 680 001 2500
Sty I C C(A,T)(A,T)GG 10 11 047 744 001 2000
Swa I ATTT AAAT 10 11 371 517 001 200
10 11 371 525 001 1000
Taq I T CGA 10 10 404 128 001 500
10 10 567 671 001 2500
10 11 175 114 001 10000
Tha I see isoschizomer Mvn I
Tru9 I T TAA 10 11 464 817 001 200
10 11 464 825 001 1000
Tth111 I see isoschizomer Asp I
Van91 I (PflM I) CCA(N)4 NTGG 5 11 379 275 001 200
Xba I T CTAGA 10 10 674 257 001 1000
10 10 674 265 001 5000
10 10 674 273 001 20000
40 11 047 663 001 20000
Xho I C TCGAG 10 10 899 194 001 5000
40 10 703 770 001 2500
40 10 703 788 001 12500
Xho II (A,G) GATC(T,C) 1­5 10 742 929 001 50
Xma III see isoschizomer EclX I
XmaC I C CCGGG 10 11 743 392 001 200
Xmn I see isoschizomer Asp 700
Volume
Activity Pack Size
Product Sequence (units/l) Cat. No. (units)
Rapid DNA Ligation of sticky-end 11 635 379 001 1 Kit
Ligation Kit or blunt-end DNA (40 DNA ligations)
fragments in just 5 min
at 15 - 25 °C.
T4 DNA Ligase Ligation of sticky- and 10 481 220 001 100 units
blunt ended DNA 10 716 359 001 500 units
fragments.
Alkaline Phos- Dephosphorylation of 11 758 250 001 1000 units
phatase, shrimp 5'-phosphate residues
from nucleic acids.
Heat inactivation:
15 min at 65 °C.
Alkaline Phos- Dephosphorylation of 11 097 075 001 1000 units
phatase (AP), 5'-phosphate residues
special quality from nucleic acids.
for molecular
biology
Agarose MP Multipurpose agarose 11 388 983 001 100 g
for analytical and pre- 11 388 991 001 500 g
parative electrophoresis
of nucleic acids.
High Pure PCR For purification of PCR 11 732 668 001 1 Kit
Product reaction products. (50 purifications)
Purification Kit
11 732 676 001 1 Kit
(250 purifications)
SuRE/Cut Incubation buffers 11 082 035 001 1 ml each
Buffer Set for A, B, L, M and H for (10 x conc. solutions)
Restriction restriction enzymes.
Enzymes
SuRE/Cut Buffer A Restriction enzyme 11 417 959 001 5 x 1 ml
incubation. (10 x conc. solution)
SuRE/Cut Buffer B Restriction enzyme 11 417 967 001 5 x 1 ml
incubation. (10 x conc. solution)
SuRE/Cut Buffer H Restriction enzyme 11 417 991 001 5 x 1 ml
incubation. (10 x conc. solution)
SuRE/Cut Buffer L Restriction enzyme 11 417 975 001 5 x 1 ml
incubation. (10 x conc. solution)
SuRE/Cut Buffer M Restriction enzyme 11 417 983 001 5 x 1 ml
incubation. (10 x conc. solution)
BSA, special Maintaining enzyme 10 711 454 001 20 mg (1 ml)
quality for stability
molecular biology
Glycogen Carrier for the precipitation 10 901 393 001 20 mg (1 ml)
of nucleic acids
(DNA or RNA)
Water, PCR Grade Specially purified, double- 03 315 932 001 25 ml
distilled, deionized, and (25 vials of 1 ml)
autoclaved water.
36
Ordering Information
Pack Size
Product Application Cat. No.
Trademarks and License Disclaimers
High Pure, Genopure, and SuRE/Cut are trademarks of a member of the Roche Group.
 This product is optimized for use in the Polymerase Chain Reaction (,,PCR") process
covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche
Ltd (,,Roche"). No license under these patents to use the PCR process is conveyed
expressly or by implication to the purchaser by the purchase of this product.
 Purchase of this product is accompanied by a limited license to use it in the
Polymerase Chain Reaction (PCR) process for life science research in conjunction with
a thermal cycler whose use in the automated performance of the PCR process is covered
by the up-front license fee, either by payment to Applied Biosystems or as purchased,
i.e., an authorized thermal cycler.
04520599990-0804
Roche Diagnostics GmbH
Roche Applied Science
68298 Mannheim
Germany