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For over 35 years, New England Biolabs has led the industry in restriction enzyme Cloning & Mapping
innovation. As the first company to commercialize restriction enzymes, we are DNA AMPLIFICATION & PCR
committed to providing a wide selection of high quality reagents at exceptional value.
RNA ANALYSIS
Our dedication to research has led to key advances in the restriction enzyme field
that allow us to offer maximum performance and convenience under a broader range Protein Expression & Analysis
of reaction conditions. Gene Expression & Cellular Analysis
Selection
In 1975, New England Biolabs (NEB) became the first company to commercialize Restriction Enzymes –
restriction enzymes, thereby providing researchers with a key set of tools that proved to be Inside the Numbers*
invaluable for the early development of the biotechnology industry. Since then, NEB
has continued an aggressive program of cloning and overexpressing restriction enzymes.
231 – restriction enzymes sold by NEB
This is done biochemically, by the analysis of cell extracts, and computationally, by the 222 – enzyme specificities
analysis of sequenced genomes. As a result, we now supply more than 220 specificities, 173 – recombinant enzymes
the largest number commercially available. 162 – enzymes exhibiting
100% activity in a single
buffer (NEBuffer 4)
153 – Time-Saver enzymes that
Quality digest substrate DNA in
5 minutes
As a result of our active screening and cloning program, NEB supplies the largest
15 – High Fidelity (HF) enzymes
engineered for reduced
selection of recombinant enzymes available. Expression using a recombinant source star activity
improves product yield and removes the uncertainties of potential contaminants. Each 10 – 8-base cutters
enzyme is carefully quality controlled using a number of sensitive assays that specifically
address nuclease contamination. This results in a higher quality product with proven
10 – nicking enzymes that cleave
only one strand of duplex DNA
lot-to-lot consistency.
4 – homing endonucleases that
r Choose recombinant enzymes for a higher quality product with lot-to-lot consistency. have large asymmetric
recognition sites
52 – Type IIS enzymes
Performance
as of 03/26/09
*
alter enzyme properties through engineering. This has 10 30 50 100 M 10 30 50 100 M 10 30 50 100
led to the discovery of nicking enzymes, the generation
of new specificities, and most recently the introduction
of a new line of High Fidelity (HF) enzymes. These engi-
neered enzymes have the same specificity as their estab-
lished counterparts, and will maximize performance under
a wider range of conditions, including reaction volume,
incubation time and buffer compatibility.
E High Fidelity enzymes have been engineered
for maximum performance.
EcoRI from other suppliers produces the correct banding pattern when 10 units are used; however, star activity is observed with larger
amounts of enzyme. Star activity is not observed with EcoRI-HF™, even at higher enzyme amounts. Reactions were set up according to
recommended reaction conditions of each manufacturer. Reactions contained 1 µg Lambda DNA in a 50 µl volume and were incubated
overnight at 37°C. Marker M is the 1 kb DNA Ladder (NEB #N3232).
2
Quality & Innovation
Convenience
• Restriction enzymes usually occur in
combination with one or two modifying
enzymes (DNA methyltransferases) that
Our dedication to restriction enzyme research has led to improvements that simplify protect the cell’s own DNA from cleavage
reaction setup. NEB utilizes a 4 buffer system, with over 160 enzymes recommended for • Type I enzymes are multisubunit proteins
use in a single buffer (NEBuffer 4). This improves efficiency and ease-of-use, especially that cut DNA randomly at a distance far
when performing double digests. Over 150 of our enzymes are Time-Saver qualified, from their recognition sequence
and will digest substrate DNA in five minutes under recommended reaction conditions. • Type II enzymes cut DNA at defined
Time-Saver qualified enzymes are easy-to-use as there is no special formulation or change positions close to or within their recogni-
in concentration. These same enzymes can be used in overnight digests without sample tion sequence and are commonly used in
loss, providing additional flexibility to reaction setup. the laboratory. There are over ten subtypes
with different types of recognition sites,
4 Over 160 restriction enzymes are recommended for use in NEBuffer 4. cleavage sites and cofactor requirements.
C Over 150 enzymes are Time-Saver qualified and will digest substrate DNA in 5 minutes. • The most common Type II enzymes
cleave within their recognition site (e.g.,
Power and purity of Time-Saver qualified restriction enzymes BamHI, EcoRI); sites can be symmetric
HindIII is powerful enough to
Incubation time (minutes) digest 1 µg of DNA in 5 minutes,
or asymmetric
0 5 15 30 60 0/N M but can also be used in overnight
digests with no indication of
• Type IIS enzymes cleave outside their
nuclease contamination. recognition sequence (e.g., FokI, AlwI)
Marker (M) is the 1 kb DNA and are invaluable for emerging technolo-
Ladder (NEB #N3232).
gies in the biotechnology industry
• Type IIM enzymes recognize methylated
targets (e.g., DpnI)
• Type III enzymes are large combination
restriction-and-modification enzymes that
cleave outside their recognition sequences
and require 2 sequences in opposite
Value orientations to cleave one DNA molecule
• Type IV enzymes recognize modified
The development of recombinant enzymes allows NEB to offer products at a lower DNA (methylated, hydroxymethylated,
cost per unit, enabling substantial savings, improved purity and consistency of product. etc.). They require two sites and cleave
There is no added cost for the convenience of Time-Saver qualified enzymes. non-specifically.
Additionally, the scientific expertise of our research staff and extensive technical • Isoschizomers are restriction enzymes
information available through our catalog and website are invaluable resources for that recognize the same sequence as
questions regarding restriction enzymes and for support in experimental design. the prototype
• Neoschizomers are isoschizomers
with different cleavage sites
www.neb.com 3
maximum performance
The following table indicates the number of units of HF-enzyme that can be used compared to the wild type counterparts
before significant star activity is detected. The HF Factor refers to the x-fold increase that is achieved by choosing an HF enzyme,
and clearly illustrates the flexibility that is offered by using an HF restriction enzyme.
Maximum Maximum
Product Product Units with no HF Product Product Units with no HF
Name Number Buffer †
Star Activity* factor Name Number Buffer †
Star Activity* factor
BamHI-HF™ #R3136 4 4,000 125 NotI-HF™ #R3189 4 + BSA 64,000 16
BamHI #R0136 3 + BSA 32 NotI #R0189 3 + BSA 4,000
EagI-HF™ #R3505 4 500 2 PvuII-HF™ #R3151 4 500 31
EagI #R0505 3 250 PvuII #R0151 2 16
EcoRI-HF™ #R3101 4 16,000 64 SacI-HF™ #R3156 4 + BSA 4,000 33
EcoRI #R0101 U 250 SacI #R0156 1 + BSA 120
EcoRV-HF™ #R3195 4 64,000 64 SalI-HF™ #R3138 4 2,000 500
EcoRV #R0195 3 + BSA 1,000 SalI #R0138 3 + BSA 4
MfeI-HF™ #R3589 4 500 15 SbfI-HF™ #R3642 4 250 31
MfeI #R0589 4 32 SbfI #R0642 4 8
NcoI-HF™ #R3193 4 16,000 133 ScaI-HF™ #R3122 4 250 62
NcoI #R0193 3 120 ScaI #R0122 3 4
NheI-HF™ #R3131 4 + BSA 32,000 266 SphI-HF™ #R3182 4 2,000 62
NheI #R0131 2 + BSA 120 SphI #R0182 2 32
SspI-HF™ #R3132 4 500
†
Wild type enzymes were tested in supplied buffer for comparisons.
SspI #R0132 U ND
* Wei, H. et al (2008) Nucleic Acids Reseach 36, e50.
4
maximum performance
References:
1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res. 14, 811.
2. Nasri, M. and Thomas, D. (1987) Nucleic Acids Res. 15, 7677.
3. Tikchonenko, T.I., et al. (1978) Gene, 4, 195.
www.neb.com 5
additional convenience
BpmI s
In an effort to provide as much information as possible, NEB has tested all of its enzymes n
on unit assay substrate as well as plasmid substrate. The rate of cutting for plasmid sub- BsaAI C l l
strate can be used as a guide as it is not definitive for all plasmids. Restriction enzymes BsaBI C l s
can often show site preference, presumably determined by the sequence flanking the BsaHI n n
recognition site. In addition, supercoiled DNA may have varying rates of cleavage. BsaWI n s
Information on site preferences and cleavage of supercoiled DNA is found in the BsaXI C l s
technical reference section of our catalog and on our website. BseRI C l l
BsgI C l l
Since all of our enzymes are rigorously tested for nuclease contamination, you can also
safely set up digests for long periods of time without sample degradation. BsiEI C l s
BsiHKAI C l n
BsiWI C l l
TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID
ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE BslI C l n
6
additional convenience
Legend:
l digests in 5 minutes Over 150 of our enzymes are Time-Saver qualified
n digests in 15 minutes and will digest 1µg of substrate DNA in 5 minutes.
s not completely digested in 15 minutes Look for the Time-Saver icon C on our website.
C Time-Saver qualified - will digest substrate DNA in 5 minutes under recommended reaction conditions
NT not tested
TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID TIME- UNIT ASSAY PLASMID
ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE ENZYME SAVER SUBSTRATE SUBSTRATE
BstUI C l l HinfI C l l PmlI C l s
www.neb.com 7
technical tips
quantity and purity. The following tips will help achieve maximum success in your Total Reaction Volume 50 µl
restriction endonuclease reactions. Incubation Time 1 hour*
BSA for optimal activity are not adversely For difficulty cleaving DNA substrate, Restriction
1 unit 5 units 10 units
Enzyme*
affected if BSA is present in the reaction we recommend the following controls:
DNA 0.2 µg 0.5 µg 1 µg
Reaction Volume • Experimental DNA without restriction
10X
enzyme to check for contamination in the NEBuffer
1 µl 2.5 µl 5 µl
• A 50 µl reaction volume is recommended DNA preparation or reaction buffer
for digestion of 1 µg of substrate 100X BSA** 0.1 µl 0.25 µl 0.5 µl
• Control DNA (DNA with multiple known
• Keep glycerol concentration at less than sites for the enzyme) with restriction
5% of total reaction volume to prevent star enzyme to test enzyme viability
* Restriction enzyme can be diluted using the recommended diluent
buffer when smaller amounts are needed.
activity † 10 µl reactions should not be incubated for longer than
• If the control DNA is cleaved and the 1 hour to avoid evaporation.
• The restriction enzyme (supplied in 50% experimental DNA resists cleavage, the ** BSA can be diluted in 1X buffer
glycerol) should not exceed 10% of the two DNAs can be mixed to determine if
total reaction volume an inhibitor is present in the experimental
sample. If an inhibitor (often salt, EDTA
or phenol) is present, the control DNA
will not cut after mixing.
8
troubleshooting
Troubleshooting Guide
Problem possible cause Solution FAQs
• Test enzyme on control DNA with known multiple sites Q. Do restriction enzymes cleave
Enzyme is inactive • Enzyme should be stored at –20°C. Enzymes stored at –70°C will
freeze, and repeated thaw/freeze cycles reduce enzyme activity. single-stranded DNA?
• Use recommended buffer supplied with restriction enzyme A. Although some restriction enzymes
Reaction conditions • Follow double digest recommendations, or try a sequential digest
are not optimal • Repeat with fresh buffer. Additives present in buffer have been reported to cleave ssDNA it
(e.g., DTT, SAM) may degrade over time. is unclear whether cleavage occurs on
Enzyme a ssDNA molecule or on two ssDNA
concentration is Some plasmids or genomic DNAs may require up to 10-20 units/µg
too low molecules which transiently anneal at a
Repeat reaction setup, being sure that enzyme and/or additives
region of partial homology (1-3). For this
Additive is missing
(e.g., BSA) are added reason, we hesitate to make unconstrained
DNA concentration NEB recommends 1 µg of DNA in a 50 µl reaction. claims about a restriction enzyme’s ability
is not optimal Excess DNA may result in incomplete cleavage. to cut ssDNA.
Incubation time is Some enzymes can exhibit slower cleavage towards specific sites.
too short In most cases, 1-2 hours are sufficient. Q. How stable are restriction enzymes?
Incomplete • Assay substrate DNA in the presence of a control DNA.
or no DNA is Control DNA will not cleave if there is an inhibitor is present. A. All restriction enzymes from NEB are
digestion contaminated with Miniprep DNA is particularly susceptible to contaminants. assayed for activity every 3-6 months.
an inhibitor • Clean DNA with a spin column, resin or drop dialysis,
or increase volume to dilute contaminant. Most are very stable when stored at
Recognition site is -20°C in the recommended storage buffer.
Confirm DNA sequence
not present Exposure to temperatures above -20°C
• DNA isolated from a bacterial source may be blocked by Dam and Dcm should be minimized whenever possible.
methylation. DNA should be passed through a dam-/dcm- strain
Cleavage is blocked (NEB #C2925).
by methylation
Q. Is extended digestion (incubation times
• Eukaryotic genomic DNA may be blocked by CpG methylation.
This can be overcome by cloning into a bacterial host. > 1 hour) recommended?
• PCR products are not methylated.
A. The unit definition of our restriction
DNA may be Restriction enzymes cleave supercoiled DNA with varying efficiency.
supercoiled Additional enzyme may be required. enzymes is based on a 1 hour incuba-
Recognition site tion. Incubation time may be shortened if
may be too close to As a general rule, add 6 base pairs on either side of the recognition site additional units of restriction enzyme are
the end of the DNA for efficient cleavage
fragment added to the reaction. Conversely, longer
Site preference Enzyme requires two recognition sites for efficient cleavage
incubation times are often used to allow
Run uncut substrate DNA alongside the digest. A partial digest will
a reaction to proceed to completion with
Determine nature of
pattern
show bands found in the uncut, whereas star activity will show bands of fewer units of enzyme. This is contingent
unexpected size. on how long a particular enzyme can
DNA sample is survive (maintain activity) in a reaction.
Unexpected Prepare a new DNA sample
contaminated
Cleavage Additional information on extended
Pattern Additional
recognition sites are Confirm DNA sequence digestion can be found at www.neb.com.
present in DNA
See tips for avoiding star activity (see page 5) and/or use a
Star Activity
High Fidelity Restriction Enzyme (see page 4)
References
Enzyme has a high
Add SDS to the gel loading dye/stop solution to a final concentration 1. Blakesley, R.W., Wells, R.D. (1975) Nature 257, 421-422.
binding affinity to
of 0.1–0.5% to help dissociate the enzyme from the DNA, or treat 2. Blakesley, R.W., et al. (1977) J. Biol. Chem. 252, 7300-7306.
DNA and will not
with protease before loading 3. Yoo, O.J., Agarwal, K.L, (1980) J. Biol. Chem. 255, 10559-
dissociate well
Smearing of 10562.
DNA on gel Nuclease Care should be taken to avoid cross contamination when
contamination setting up reactions
Agarose running
Use fresh running buffer and appropriate voltage to avoid overheating
conditions
www.neb.com 9
technical reference
Digesting DNA substrate with two restriction endonucleases simultaneously (double digestion)
is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions
that optimize enzyme activity and avoid star activity associated with some enzymes is
an important consideration. Each enzyme is supplied with an NEBuffer that ensures 100%
activity. (For compositions, see page 15). The Activity Chart for Restriction Enzymes
(pages 15 - 19) rates the percentage activity of each restriction endonuclease in the four
standard NEBuffers and NEBuffer EcoRI.
Setting up a Double Digest • If two different incubation temperatures Additional double digest information with
are necessary, choose the optimal reac- unique buffers can be found on our website,
• Choose an NEBuffer that results in the www.neb.com.
tion buffer and set up reaction accordingly.
most activity for both enzymes, provided Add the first enzyme and incubate at the
that star activity is not a concern. desired temperature. Heat inactivate the first Setting up a Sequential Digest
• If BSA is required for either enzyme, enzyme, add the second enzyme and • Set up a reaction using the restriction
add it to the double digest reaction (BSA incubate at the recommended temperature. endonuclease that has the lowest salt
does not inhibit restriction enzymes). concentration in its recommended buffer
• Depending on an enzyme’s activity rating
• Set up reaction according to recom- and incubate to completion.
in a non-optimal NEBuffer, the number of
mended conditions (see p. 8). The final • Adjust the salt concentration of the
units or incubation time may be adjusted
concentration of glycerol in any reaction reaction (using a small volume of a
to compensate for slower rate of cleavage.
should be less than 5% to minimize the concentrated salt solution) to approxi-
possibility of star activity (see p. 5). Setting up a Double Digest mate the reaction conditions of the
For example, in a 50 µl reaction, the second restriction endonuclease.
total amount of enzyme added should with a unique buffer
• Add the second enzyme and incubate
not exceed 5 µl. • Our buffer system has been streamlined, to complete the second reaction.
• Incubate at recommended temperature. leaving three enzymes that have unique
• Alternatively, a spin column can be used
Overnight digests should be avoided due NEBuffers: EcoRI (included in activity
to isolate the DNA prior to the second
to the possibility of star activity. chart), SspI (same buffer composition as
reaction.
EcoRI) and DpnII. In most cases, DpnII
requires a sequential digest.
Double Digest Chart
ENZYME AatII AvrII BamHI BglII BsgI EagI EcoRI EcoRV HindIII KpnI MseI NcoI NdeI NheI NotI PstI PvuI SacI SacII SalI SmaI SpeI SphI XbaI XhoI
NEBuffer 4 4 3 3 4 3 U 3 2 1 4 3 4 2 3 3 3 1 4 3 4 4 2 4 4
AvrII 4 4
BamHI 3 seq 3
BglII 3 seq 2 3
BsgI 4 4 4 3 3 Enzymes in purple are also available in
EagI 3 seq 3 3 3 seq a High Fidelity (HF) format. HF enzymes
EcoRI U seq EcoRI EcoRI EcoRI seq EcoRI are 100% active in NEBuffer 4 and will
EcoRV 3 4 2 3 3 4 3 EcoRI simplify double digest reactions.
HindIII 2 4 2 seq 2 2 seq seq 2
KpnI 1 4 1 seq 2 seq seq 1 2 2
MseI 4 4 4 3 2 4 3 EcoRI 2 2 1
NcoI 3 4 4 3 3 4 3 EcoRI 3 2 1 4
NdeI 4 4 4 3 3 4 3 EcoRI 2 2 1 4 4
NheI 2 4 2 seq 2 4 seq 1 2 2 1 2 2 4
NotI 3 seq 3 3 3 3 3 EcoRI 3 2 2 2 3 3 2
PstI 3 4 3 3 3 3 3 EcoRI 3 2 1 3 3 3 2 3
PvuI 3 seq 2 3 3 3 3 EcoRI 3 2 2 3 3 3 2 3 3
SacI 1 4 1 seq 2 4 seq 1 2 2 1 4 1 4 1 2 1 2
SacII 4 4 4 seq seq 4 seq EcoRI 2 2 4 4 4 4 4 2 2 2 4
SalI 3 seq 3 3 3 3 3 EcoRI 3 seq seq 3 3 3 seq 3 3 3 seq seq
SmaI 4 4 4 seq seq 4 seq seq 4 4 seq 4 4 4 4 seq 4 seq 4 4 seq
SpeI 4 4 4 seq 2 4 seq EcoRI 2 2 1 4 4 4 2 2 2 2 1 4 seq 4
SphI 2 4 2 3 2 4 3 EcoRI 2 2 1 2 2 2 2 2 2 2 1 4 3 4 2
XbaI 4 4 4 3 2 4 3 seq 2 2 2 4 4 4 2 3 3 3 4 4 3 4 4 2
XhoI 4 4 4 3 3 4 3 EcoRI 3 2 1 4 4 4 2 3 3 3 4 4 3 4 4 2 4
XmaI 4 4 4 seq seq 4 seq seq 4 seq 4 4 4 4 4 2 4 seq 4 4 seq 4 4 4 4 4
10
technical reference
• Mammalian and plant DNA that has been cloned into a methylating E. coli strain will be
Dam/Dcm methylated. Most commonly used laboratory E. coli strains methylate DNA.
• Directly isolated mammalian and plant genomic DNA are CpG methylated. Some enzymes
are inhibited by CpG methylation. (See www.neb.com for more information).
• Most bacterial DNA (including E. coli DNA) is not CpG methylated. Inhibition of enzyme
activity by CpG methylation is not an issue for DNA prepared from E. coli strains.
• DNA amplified by PCR does not contain any methylated bases.
• To avoid Dam/Dcm methylation when subcloning in bacteria, NEB offers the
methyltransferase deficient cloning strain dam–/dcm– Competent E. coli (NEB #C2925)
for propagation.
www.neb.com 11
web tools
Online Tools
As the leader in enzyme technology, NEB’s technical support is an invaluable resource.
Our scientists provide assistance via info@neb.com or 1-800-NEB-LABS (1-800-632-7799).
To aid in experimental design, NEB has created several user-friendly web-based online tools.
These tools and the technical reference section of www.neb.com can help you choose an enzyme,
design an experiment or troubleshoot an unexpected result.
Enzyme Finder
Enzyme Finder can be used to search for
restriction enzymes by name, sequence, overhang
or type. Search results include all enzyme matches,
with properties for NEB enzymes listed.
www.neb.com/nebecomm/EnzymeFinder.asp
NEBcutter
NEBcutter allows you to input sequence data and find large
ORFs, identify restriction sites and generate custom digests
and virtual gels. Sequences submitted are maintained locally
for 2 days and then discarded for your privacy.
tools.neb.com/NEBcutter2/index.php
Submit your sequence to find largest
ORF, identify restriction sites and
generate custom digests
Zoom in to view
cleavage overhangs
REBASE
REBASE (The Restriction Enzyme Database) is a complete listing of all known restriction
endonucleases. It contains data such as recognition sequences, cleavage sites, methylation
sensitivity, isoschizomers and commercial availability of enzymes.
rebase.neb.com/rebase/
Click here for specialized infor- Access available tools (theoretical digests,
mation (star activity, methylation, BLAST, NEBcutter, REBpredictor)
cleavage type, etc.), REBASE
genomes and sequence collections
www.neb.com 13
technical reference
Experimental: Linearized vectors were incubated with the indicated enzymes (10 units/µg) The technical reference section of our website
for 60 minutes at the recommended reaction conditions for each enzyme. Following ligation contains additional charts, protocols and
and transformation, cleavage efficiencies were determined by dividing the number of technical tips related to restriction enzymes.
transformants from the digestion reaction by the number obtained from religation of the
linearized DNA (typically 100–500 colonies) and subtracting from 100%. “Base Pairs
from End” refers to the number of double-stranded base pairs between the recognition
site and the terminus of the fragment; this number does not include the single-stranded
overhang from the initial cut.
14
selection
Note: The values listed in this table are approximate. Values were obtained using each enzyme’s specific unit assay substrate DNA. Updates to this chart can
be found on our web site, www.neb.com.
www.neb.com 15
selection
HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE
16
selection
HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE
www.neb.com 17
selection
HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE
HEAT
SUPPLIED % ACTIVITY in NEBuffers INACTIVATION INCUBATION
ENZYME NEBuffer 1 2 3 4 EcoRI (TEMP) TEMPERATURE
info@ca.neb.com BOSTON, MA
PERMIT NO. 54302
China, People’s Republic New England Biolabs, Inc.
New England Biolabs (Beijing), Ltd. 240 County Road
Telephone: 010-82378265/82378266 Ipswich, MA 01938-2723
info@neb-china.com
Germany
New England Biolabs GmbH
Free Call: 0800/246 5227
info@de.neb.com
United Kingdom
New England Biolabs (UK), Ltd.
Call Free 0800 318486
info@uk.neb.com
Japan
New England Biolabs Japan, Inc.
Telephone: +81 (0)3 5669 6191
info@neb-japan.com
www.neb.com
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