Download EPICENTRE Enzyme Catalog

Enzyme Catalog
Your trusted source for specialty molecular biology enzymes
EPICENTRE Profile
Introduction
Our expertise in producing enzymes
Unit definition
Animal Product Free
EPICENTRE Biotechnologies develops,
manufactures, and sells high-quality enzyme
systems for life science research. Located in
Madison, Wisconsin, EPICENTRE was founded
in 1987, and now occupies a state-of-the art
72,000-s.f. building. EPICENTRE is well-known
for its unique expertise in making a broad
range of enzymes for molecular biology, many
of which are unique to EPICENTRE. EPICENTRE
products are available directly in the United
States and internationally through authorized
distributors
A significant number of commercially available
enzymes have different unit definitions from
different suppliers. Unit definition is directly
related to the protocol on how to use an enzyme
and to its actual cost. Some of our enzymes’ unit
definition makes one EPICENTRE unt equivalent
to multiple units of the same enzymes from
some other suppliers. Please visit www.EpiBio.
com or call our Tech Support at 1-800-2848474 if you have questions on our enzyme unit
definition.
We also produce a range of animal productfreeenzymes. Please visit www.EpiBio.com or
call Customer Service at 1-800-274-8474 for
more information
Highest enzyme purity in the market
EPICENTRE’s optimal and proprietary enzyme
production procedure ensures that no tags
are used in the production of our final enzyme
products. If you are concerned about various
tags affecting your research applications,
EPICENTRE is your choice of enzyme source.
EPICENTRE takes pride in its optimized and
proprietary protein production, purification and
quality control procedures that contribute to the
extremely high purity of our enzymes products.
Tag free
Bulk and custom offerings
EPICENTRE always tries to accommodate
our customers’ needs for your specific
applications. If you need enzymes with bulk
quantitities, and/or custom formulations (e.g.
higher concentrations), please inquire with our
Customer Service at 1-800-274-8474.
Limited use license
EPICENTRE’s products are labeled for research
or laboratory use only. For any orther use please
inquire through our toll free number 1-800-2848474.
NOTICE TO PURCHASER: LIMITED LICENSE
*Use of MasterAmp™ AmpliTherm™ DNA Polymerase, MasterAmp™ Taq DNA Polymerase, MasterAmp™ Tfl DNA Polymerase, or MasterAmp™ Tth DNA
Polymerase is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711,
6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity
from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent
claim (such as the patented 5´ Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no
right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial
consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a
separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850
Lincoln Centre Drive, Foster City, California 94404, USA.
Use of MasterAmp™ PCR Enhancer DNA Polymerase Reactions, including, but not limited to use for PCR or DNA Sequencing, is covered by U.S.
Patent No. 6,270,962, European Patent No. 0742838, German Patent No. DE4411588C1, and other issued or pending applications in the U.S. and other
countries that are either assigned or exclusively licensed to EPICENTRE. These products are accompanied by a limited non-exclusive license for the purchaser
to use the purchased products solely for life science research. Contact EPICENTRE for information on licenses for uses in diagnostics or other fields.
2
[email protected] • www.EpiBio.com
T7, T3, and SP6 RNA Polymerases
• Extremely high promoter specificity.
Applications
•RNA synthesized can be used as a
hybridization probe, anti-sense RNA, a
ribozyme, a template for in vitro translation,
as a precursor mRNA for splicing or other
processing studies, or to make dsRNA for
RNA interference or gene silencing.
•Synthesis of RNA for nucleic acid
amplification methods or gene expression
studies.
Catalog No.
Conc.
T7 RNA Polymerase
T7905K
50 U/µl
T7925K
50 U/µl
TM910K
200 U/µl
TM925K
200 U/µl
TH950K
1,000 U/µl
TU950K
2,500 U/µl
Size
Price $
5,000 U
25,000 U
10,000 U
25,000 U
50,000 U
50,000 U
(Enzyme only. Transcription Buffer is not included.)
T3 RNA Polymerase
T4905K
50 U/µl
T9050K
50 U/µl
TM4910K
200 U/µl
TH4950K
1,000 U/µl
5,000 U
50,000 U
10,000 U
50,000 U
(Enzyme only. Transcription Buffer is not included.)
SP6 RNA Polymerase
S4905K
50 U/µl
S4950K
50 U/µl
SM910K
200 U/µl
5,000 U
50,000 U
10,000 U
(Enzyme only. Transcription Buffer is not included.)
RNA Polymerases and Replicases
Produce defined RNA by in vitro transcription of
double-stranded DNA that is downstream of the
respective RNA polymerase promoter.
Transcription Buffer Package
BP1001
1 pkg
Includes 5 ml of 5X Transcription Buffer and 2.5 ml of 100
mM DTT.
*Greatest range of enzyme concentrations available from 50
U/µl to 2,500 U/µl.
**For kits for in vitro transcription, please visit
www.EpiBio.com/ivt.asp.
T7 R&DNA™ Polymerase and SP6 R&DNA™ Polymerase
Recombinant enzymes having single-base
active-site mutations in the respective T7 or
SP6 RNA Polymerase gene. These active-site
mutations enable the corresponding T7 or
SP6 R&DNA Polymerase to incorporate 2’deoxyribonucleoside triphosphates (dNTPs) into
full-length transcripts much more efficiently
than the corresponding wild-type enzymes,
while retaining the same catalytic activity for
incorporation of canonical NTPs and the same
high promoter specificity.
Applications
•Synthesis of “RNA” transcripts of mixed
rNMP/2’-dNMP or rNMP/2’-modified-NMP
composition.
•Synthesis of modified “RNA” transcripts that
are resistant to RNase A-type RNases.
T7 & SP6 R&DNA™ Polymerases, & DuraScribe™ T7 & SP6
Transcription Kits to synthesize nucleic acids with non-canonical
bases or for partial ribosubstitution are covered by U.S. Patents
5,849,546; 6,107,037; or 6,596,494, and other patents issued
or pending. These products are accompanied by a limited nonexclusive license for the purchaser to use the purchased product(s)
solely for life science research except the development of
therapeutics. Contact EPICENTRE concerning licenses for other uses.
Catalog No.
Conc.
T7 R&DNA™ Polymerase
D7P9201K
50 U/µl
D7P9205K
50 U/µl
Size
Price $
1,000 U
5,000 U
Contents: T7 R&DNA™ Polymerase, 5X Reaction Buffer, and
100 mM DTT.
SP6 R&DNA™ Polymerase
D6P9301K
50 U/µl
D6P9305K
50 U/µl
1,000 U
5,000 U
Contents: SP6 R&DNA™ Polymerase, 5X Reaction Buffer,
and 100 mM DTT.
*For kits for in vitro transcription, please visit
www.EpiBio.com/ivt.asp.
E. coli RNA Polymerase Core Enzyme and Sigma-Saturated Holoenzyme
Both enzyme preparations are isolated from the
rifampicin-sensitive strain BL21. EPICENTRE
is the only company that offers purified Core
Enzyme, which has no detectable sigma subunit,
and 100% Sigma-Saturated (σ70)-Holoenzyme.
Applications
•The Core Enzyme is useful in studying
mechanisms of transcription initiation,
since it will not initiate specific transcription
at promoter sequences on bacterial or
bacteriophage DNA due to a lack of sigma
factor.
•The sigma-saturated Holoenzyme is very
efficient in specifically transcribing a variety
of double-stranded DNA templates containing
promoters.
Holo Core
- β´, β
-σ
-α
-ω
FIG 1. Subunit patterns of
E. coli RNA Polymerase
Core Enzyme and
Holoenzyme preparations.
Equivalent amounts of each
enzyme were separated by
electrophoresis on an SDS
15% polyacrylamide gel,
stained with Coomassie Blue,
and dried. The sigma-70
subunit is 100% saturating in
the Holoenzyme, but is absent
in the Core Enzyme.
Catalog No.
Conc.
Size
E. coli RNA Polymerase Core Enzyme
C90100
100 U
C90250
250 U
E. coli RNA Polymerase Holoenzyme
(Sigma-Saturated)
S90050
50 U
S90100
100 U
*For kits for in vitro transcription, please visit
www.EpiBio.com/ivt.asp.
Thermus Thermostable RNA Polymerase
Derived from the thermophilic bacterium,
Thermus thermophilus, this RNA Polymerase
is the only commercially-available RNA
www.EpiBio.com • [email protected]
polymerase that is stable and has optimal
activity at temperatures above 65°C.
Catalog No.
Conc.
Size
Price $
Thermus Thermostable RNA Polymerase
T90050
50 U
T90100
100 U
3
RNA Capping and Tailing Enzymes
ScriptCap™ m7G Capping System
Catalog No.
Conc.
Size
ScriptCap™ m7G Capping System
SCCE0610
- 10 Reactions
SCCE0625
- 25 Reactions
Contents: ScriptCap™ Capping Enzyme, 10X Capping Buffer,
ScriptGuard™ RNase Inhibitor, 20 mM SAM, 10 mM GTP, and
RNase-Free Water.
*For complete kits designed to produce capped and tailed
RNA in vitro, please visit www.EpiBio.com/capping.asp.
Based upon the tri-functional Vaccinia Virus
capping enzyme (VCE), this system is designed
to build the Cap 0 structure found on the 5´
end of most eukaryotic mRNA molecules. This
enzyme system is identical to the Vaccinia
guanylyltransferase sold by other vendors.
Applications
• In vitro production of capped RNA for in vivo
or in vitro translation.*
•Analysis of 5´ ends of RNA transcripts.
Figure: (A) Denaturing Polyacrylamide gel and (B)
autoradiograph of a ScriptCap™ Capping system
reaction. Lane 2 shows the uncapped transcript
without Vaccinia capping enzyme (VCE). Lane
3 includes the VCE and 14C SAM. VCE has clearly
transferred the guanine base (Lane 3A) and the 14C
containing methyl group (Lane 3B).
ScriptCap™ 2’-O-Methyltransferase
Catalog No.
Conc.
Size
ScriptCap™ 2’-O-Methyltransferase
SCMT0610
- 10 Reactions
SCMT0625
- 25 Reactions
Contents: ScriptCap™ 2’-O-Methyltransferase, 10X Capping
Buffer, and 20 mM SAM.
* For complete kits designed to produce capped and tailed
RNA in vitro, please visit www.EpiBio.com/capping.asp.
The ScriptCap 2’-O-Methyltransferase
Enzyme is derived from the Vaccinia virus and
methylates the penultimate nucleotide in a
capped eukaryotic mRNA transcript, converting
a Cap 0 transcript into the natural Cap 1 mRNA
structure. This methylation results in an up to
50% increase in the in vivo translation efficiency
when compared to Cap 0 mRNA.
The enzyme is active on both the natural
Cap 0 structure and several different Cap 0
dinucleotide analogs used in co-transcriptional
capping kits. Each reaction is capable of
methylating 60 ug of Cap 0 RNA in 30 minutes.
Applications
Table 1. Various different forms of capped and tailed mRNA were transfected into HeLa cells and assayed
for luciferase activity. Data is normalized to the Cap 0, poly(A) transfection results. The complete Cap 1 mRNA
exhibited up to 50% higher in vivo translation efficiency when compared to the various Cap 0-type structures
currently used in most transfections.
Methylation of Cap 0 RNA to the Cap 1 form
Means of Cap 0 Production
ScriptCap™
Final mRNA Cap
2’-O-Methyltransferase
Structures Formed
Treatment
No RNA
no
none
ScriptCap™ m7G Capping System
no
m7GpppN
(Capping Enzyme)
(Cap 0)
ScriptCap™ m7G Capping System
yes
m7Gppp[m2’-O]N
(Capping Enzyme)
(Cap 1)
AmpliCap-Max™ High Yield
no
m7GpppN *
Message Maker Kit
(Cap 0)
(Standard Cap Analog)
AmpliCap-Max™ High Yield
yes
m7Gppp[m2’-O]N *
Message Maker Kit
(Cap 1)
(Standard Cap Analog)
AmpliScribe™ T7-Flash™
no
m27, 3´-OGpppN *
Transcription Kit
(Cap 0)
(ARCA Cap Analog)
AmpliScribe™ T7-Flash™
yes
m27, 3´-OGppp[m2’-O]N *
Transcription Kit
(Cap 1)
(ARCA Cap Analog)
Translation Efficiency
relative to non-treated
mRNA
0%
100%
147%
100%
148%
100%
128%
A-Plus™ Poly(A) Polymerase
Catalog No.
Conc.
Size
A-Plus™ Poly(A) Polymerase Tailing Kit
50
PAP5104H
4 U/µl
Reactions
(400 U)
Contents: A-Plus™ Poly(A) Polymerase, A-Plus™ 10X
Reaction Buffer, 10 mM ATP, and Sterile RNase-Free Water.
A-Plus™ Poly(A) Polymerase uses ATP as a
substrate for template-independent addition of
adenosine monophosphate to the 3´-hydroxyl
termini of RNA molecules. The A-Plus™ Poly(A)
Polymerase Tailing Kit provides the enzyme
and other reagents to quickly and easily add a
“poly(A) tail” to the 3´-end of any RNA.
Applications
•Addition of a poly(A) tail to RNA synthesized
in vitro to increase RNA stability and enhance
its in vivo translation after transfection or
microinjection into eukaryotic cells.
• Addition of a poly(A) tail to RNA molecules to
4
provide a priming site for synthesis of firststrand cDNA using a primer with poly(dT) on
its 3´-end.
•Cloning of DNA encoding RNA molecules of
unknown or multiple sequences by adding
a poly(A) tail that can anneal to a T-tailed
vector.
•Synthesis of polyadenylated RNA for
nucleic acid amplification methods or gene
expression studies.
•3´-End-labeling of RNA with radioactive A
residues.
•Quantifying mRNA.
[email protected] • www.EpiBio.com
Properties of Mesophilic DNA Polymerases
3´→5´ exonuclease
Activity
Nick translation
Heat Inactivationa
Strand displacement
DNA Polymerase I, E. coli
+
++
+
75°C 20 minutes
-
Klenow DNA Polymerase
-
++
-
75°C 20 minutes
+
Exo-Minus Klenow DNA
Polymerase
-
-
-
75°C 20 minutes
+
RepliPHI™ Phi29 DNA
Polymerase
-
++
-
65°C 10 minutes
++++
T4 DNA Polymerase
-
+++
-
75°C 20 minutes
-
T7 DNA Polymerase,
Unmodified
-
+++
-
75°C 20 minutes
-
a
DNA Polymerases
5´→3´ exonuclease
Product Name
Indicated treatment results in complete inactivation under standard reaction conditions
DNA Polymerase I, E. coli
This DNA-dependent DNA polymerase contains
both 5’→3’and 3´→5´ exonuclease activities.
The 5´→3´ exonuclease activity enables the
enzyme to use nicks and gaps in the DNA as
starting points for labeling the DNA by nick
translation.
Applications
•Generate labeled DNA probes by nick
translation.
•Second strand cDNA synthesis
Catalog No.
Conc.
DNA Polymerase I, E. coli
DP02250
10 U/µl
DP021K
10 U/µl
Size
250 U
1000 U
• In vitro synthesis of DNA.
Klenow DNA Polymerase
Derived from E. coli DNA polymerase I, this
large fragment, DNA-dependent enzyme has
5´→3´ polymerization and 3´→5´ exonuclease
activities, but lacks 5´→3´ exonuclease
activity. Klenow DNA polymerase blunt ends
doublestranded DNA with singlestranded
overhangs. The 3´→5´ exonuclease activity
removes 3´ overhangs and the 5´→3´
polymerization activity fills in 5´ overhangs.
Applications
• Random primer labeling of DNA.
• DNA sequencing.
Catalog No.
Conc.
Klenow DNA Polymerase
KP04061K
-
Size
1000 U
• Second-strand cDNA synthesis.
• Strand displacement amplification.
Exo-Minus Klenow DNA Polymerase
This DNA-dependent DNA polymerase lacks
both the 5´→ 3´ and 3´→5´ exonuclease
activities of E. coli DNA Polymerase I from which
it is derived.
Applications
• Random primer labeling of DNA.
• DNA sequencing.
Catalog No.
Conc.
Size
Exo-Minus Klenow DNA Polymerase
KL04011K
20 U/µl 1000 U
KL06041K
50 U/µl 1000 U
• Second-strand cDNA synthesis.
• Strand displacement amplification.
RepliPHI™ Phi29 DNA Polymerase
RepliPHI™ Phi29 DNA Polymerase (φ29 DNA
Polymerase) is a highly processive enzyme with
exceptional strand displacement activity. The
www.EpiBio.com • [email protected]
enzyme also contains a 3´→5´ exonuclease
activity that enables proofreading capability. Its
specific activity is 1 x 106 U/mg.
Catalog No.
Conc.
Size
RepliPHI™ Phi29 DNA Polymerase (enzyme only)
10 µg
1 µg/µl
PP031010
(1,000 U/µl)
(10,000 U)
10 µg
0.1 µg/µl
PP040110
(100 U/µl)
(10,000 U)
RepliPHI™ Phi29 Reagent Set
(includes enzyme, buffer, dNTPs, DTT)
Enzyme:
Enzyme:
RH031110
1 µg/µl
10 µg
(1,000 U/µl)
(10,000 U)
Enzyme:
Enzyme:
RH040210
0.1 µg/µl
10 µg
(100 U/µl)
(10,000 U)
RepliPHI™ Phi29 Polymerase Dilution Buffer
RPB04041
1 ml
5
DNA Polymerases
T4 DNA Polymerase
Catalog No.
Conc.
T4 DNA Polymerase
D0602H
D0605H
-
Contains both a template-directed 5´→3´
DNA polymerase activity and a potent 3´→ 5´
exonuclease activity.
Size
200 U
500 U
Applications
•Conversion of 5´- and 3´-protruding DNA
termini to blunt ends.
•Cloning of PCR fragments: Treatment of PCR
products containing 3´-A overhangs with T4
DNA Polymerase and dNTPs produces blunt
ends.
•Production of site-specific mutations:
this enzyme can be used for site-specific
mutagenesis by primer extension of
“mutated” oligonucleotides hybridized to
single-stranded DNA templates.
•Labeling of 3´-termini of DNA molecules and
synthesis of strand-specific probes using the
exonuclease and polymerase activities of T4
DNA Polymerase.
T7 DNA Polymerase, Unmodified
Contains both a template-directed 5´→ 3´
DNA polymerase activity and a potent 3´→
5´ exonuclease activity. The unmodified form
of T7 DNA Polymerase is different from T7
DNA polymerase preparations from which
exonuclease activity has been removed. The
enzyme is a tightly-bound complex of T7
phage-encoded gene 5 protein and E. coli hostencoded thioredoxin.
Catalog No.
Conc.
Size
T7 DNA Polymerase, Unmodified
D07250
10 U/µl
250 U
D07500
10 U/µl
500 U
Highly processive and synthesizes long
stretches of DNA before dissociating from the
template. The rate of elongation is also much
faster than that of most other DNA polymerases.
Applications
•Primer extension of long DNA molecules.
•Conversion of 5´- and 3´-protruding ends to
blunt ends.
•Labeling of 3´-ends of DNA.
• In situ detection of DNA fragmentation
associated with apoptosis.
Properties of Thermophilic DNA Polymerases
Product Name
Activity
Thermostabilityb
Fidelityc
Strand
displacement
<15 min at 75°C
<1.5 min at 95°C
N/A
-
-
<15 min at 75°C
<1.5 min at 95°C
N/A
+++
-
-
N/A
N/A
N/A
Very weak.
Requires
Mn2+
+
-
9 min at 97.5°C
0.38-1.82 x 104
N/A
MasterAmp™ Tfl DNA Polymerase
-
+
-
40 min at 95°C
8.3-9.0 x 105
N/A
MasterAmp™ Tth DNA Polymerase
++
Requires
Mn2+
+
-
20 min at 95°C
2.2 x 104
N/A
Reverse
transcriptase
5´→3´
exonuclease
3´→5´
exonuclease
rBst DNA Polymerasea
+
+
-
rBst DNA Polymerase Large
Fragment, (IsoTherm™ DNA
Polymerase)a
-
-
MasterAmp™ AmpliTherm™
DNA Polymerase
-
MasterAmp™ Taq DNA Polymerase
a rBst DNA polymerase and rBst DNA Large Fragment (IsoTherm™ DNA Polymerase) are for DNA replication at < 65°C. Not suitable for typical PCR. The other thermostable enzymes listed in this chart are
suitable for PCR cycling applications.
b Values represent half-lives: 50% of the enzymatic activity is retained after the given time at the stated temperature.
c Defined as the average number of correct nucleotides a polymerase incorporates before making an error.
rBst DNA Polymerase
Catalog No.
Conc.
rBst DNA Polymerase
BH1100
5 U/µl
BH1500
5 U/µl
Size
100 U
500 U
Derived from the DNA pol I gene of
the thermophilic bacterium Bacillus
stearothermophilus (Bst), this enzyme has optimal
activity at 65°C and at elevated temperatures
it can synthesize DNA in regions containing
template secondary structure or high GC content
where other non-thermostable DNA polymerases
may fail. It has 5´→ 3´ exonuclease activity.
Its optimal activity as elevated temperature
6
makes it useful for replicating difficult
templates.
Applications
•High temperature DNA synthesis.
•First strand cDNA synthesis from primed RNA
templates.
•Production of cDNA templates for use in
amplification reactions.
[email protected] • www.EpiBio.com
rBst DNA Polymerase, Large Fragment (IsoTherm™ DNA Polymerase)
Its high rate of DNA synthesis permits use
of nanogram quantities of template under
isothermal conditions.
Ability to synthesize through regions of high GC
content or difficult secondary structure at high
temperatures.
•High-temperature isothermal DNA
sequencing.
Applications
•Amplification assays and other assays
involving continuous displacement of the DNA
strand concomitant with high-temperature
DNA synthesis.
Catalog No.
Conc.
Size
rBst DNA Polymerase, Large Fragment
(IsoTherm™ DNA Polymerase)
BL901K
5 U/µl
1,000 U
BL1805K
50 U/µl
5,000 U
BL1950K
50 U/µl 50,000 U
Enzyme only.
•First-strand cDNA synthesis from primed RNA
templates.
DNA Polymerases
Derived from the DNA pol I gene of
the thermophilic bacterium Bacillus
stearothermophilus (Bst) altered to remove the
5´→ 3´ DNA exonuclease activity. Having optimal
DNA polymerase activity at 65°C, the enzyme
is suitable for high-temperature isothermal
sequencing of DNA. Like the Klenow fragment
of E. coli DNA Polymerase I, this enzyme has
strong strand displacement activity. It also has
thermostable RNA-dependent DNA polymerase
(i.e., reverse transcriptase) activity.
MasterAmp™ AmpliTherm™ DNA Polymerase*
This proprietary recombinant thermostable
DNA polymerase for use in PCR has optimal
DNA synthetic activity at temperatures above
70°C and can be used at temperatures up to
95°C. It lacks both 5´→ 3´ structure-dependent
exonuclease activity like that found in Taq
DNA polymerase and 3´→ 5´ proofreading
exo- nuclease activity found in some other DNA
polymerases.
Provided with the MasterAmp™ PCR Enhancer,
which increases the probability of obtaining the
desired amplification product, the reproducibility
of PCR, and improves the consistency of PCR
product yields in multiplex PCR.
Applications
• PCR amplification of DNA templates.
• Multiplex PCR.
Catalog No.
Conc.
Size
MasterAmp™ AmpliTherm™ DNA Polymerase
AT72250
5 U/µl
250 U
Includes 10X PCR Buffer, 25 mM MgCl2, and MasterAmp™
10X PCR Enhancer.
MasterAmp™ AmpliTherm™ DNA Polymerase
(Enzyme Only)
AT72250N
5 U/µl
250 U
For use with MasterAmp™ PCR PreMixes.
MasterAmp™ Taq DNA Polymerase*
This thermostable DNA polymerase derived
from Thermus aquaticus has optimal activity
at temperatures above 70°C. It has an intrinsic
5´→ 3´ structure-dependent exonuclease
activity, but lacks 3´→ 5´ proofreading
exonuclease activity. Reliable activity, specificity,
and reproducibility in PCR in conjunction with
the MasterAmp PCR Enhancer Technology.
Applications
•PCR and Multiplex PCR amplification of DNA
templates.
Catalog No.
Conc.
Size
MasterAmp™ Taq DNA Polymerase
Q82250
5 U/µl
250 U
Q8201K
5 U/µl
1,000 U
Includes 10X PCR Buffer, 25 mM MgCl2, and MasterAmp™
10X PCR Enhancer.
MasterAmp™ Taq DNA Polymerase (Enzyme Only)
Q82250N
5 U/µl
250 U
For use with MasterAmp™ PCR PreMixes.
MasterAmp™ Taq PCR Core Kit
200 Reactions
MCQ74200
Contents: MasterAmp™ Taq DNA Polymerase, 10X PCR
Buffer, MasterAmp™ 10X PCR Enhancer, dNTP Mix, 2.5
mM each, 25 mM MgCl2, Enzyme Dilution Buffer, Control
Template and Primers Mix.
MasterAmp™ Tfl DNA Polymerase*
Derived from the thermophilic bacterium Thermus
flavus, this is a recombinant DNA polymerase
with good thermostability (to ~95°C) and
processivity (with 15 kb PCR products reported).
MasterAmp™ PCR Enhancer Technology
substantially increases the probability of
obtaining the desired amplification product and
the reaction-to-reaction consistency, and greatly
improves the consistency of PCR product yields in
multiplex PCR.
Applications
•PCR and Multiplex PCR amplification of DNA
templates.
•Produces PCR products up to 15 kb.
•Adaptable to high-throughput PCR formats.
Catalog No.
Conc.
Size
MasterAmp™ Tfl DNA Polymerase
F72250
1 U/µl
250 U
F7201K
1 U/µl
1,000 U
Includes 20X PCR Buffer, 25 mM MgCl2, and MasterAmp™
10X PCR Enhancer.
MasterAmp™ Tfl DNA Polymerase (Enzyme Only)
F72250N
1 U/µl
250 U
For use with MasterAmp™ PCR PreMixes.
MasterAmp™ Tth DNA Polymerase*
This recombinant DNA enzyme from Thermus
thermophilus has both DNA-dependent and
RNA-dependent (i.e., reverse transcriptase) DNA
polymerase activities up to ~95°C. High reaction
temperatures can reduce nonspecific priming
and template secondary structure. Provided with
MasterAmp™ PCR Enhancer Technology.
dependent DNA polymerase activity under the
same reaction conditions.
Has both reverse transcriptase and DNA-
•1-step RT-PCR of RNA templates.
Improved PCR of RNA and DNA templates having
a high degree of secondary structure.
Applications
•PCR amplification of DNA
Catalog No.
Conc.
Size
MasterAmp™ Tth DNA Polymerase
TTH72100
5 U/µl
100 U
TTH72250
5 U/µl
250 U
TTH7201K
5 U/µl
1,000 U
Includes a 20X PCR Buffer (without Mg2+ or Mn2+) plus
separate 25 mM solutions of MgCl2 and MnSO4, and
MasterAmp™ 10X PCR Enhancer.
MasterAmp™ Tth DNA Polymerase (Enzyme Only)
TTH7225N
5 U/µl
250 U
For use with MasterAmp™ PCR PreMixes.
www.EpiBio.com • [email protected]
*Refer to page 2 for patent and license information.
7
Catalog No.
Conc.
Size
MMLV Reverse Transcriptase 1st-Strand
cDNA Synthesis Kit
MM070110
10 Reactions
MM070150
50 Reactions
Contents: MMLV Reverse Transcriptase, 10X Reaction
Buffer, ScriptGuard™ RNase Inhibitor, dNTP PreMix, DTT,
Oligo(dT)21Primer, Random 9-mer Primers, RNase-free
Water.
*See page XX for RNase H and related products
The MMLV Reverse Transcriptase 1st-Strand
cDNA Synthesis Kit is optimized for generating
full-length first-strand cDNA from total cellular
RNA preparations or purified polyadenylated RNA.
•Synthesize full-length cDNA from RNA
templates longer than 15 kb.
~ 15.2 kb HERC1 mRNA
5'
1A
AAAA(A)n 3'
cDNA Synthesis
3'
TTTT(T)17 5'
1.3 kb PCR product
•Both oligo(dT) and random primers included
in the kit.
i2130703
Reverse Transcriptase
MMLV Reverse Transcriptase
1B
•The kit includes both an oligo(dT)-containing
and a random nonamer (9-mer) primer.
•First-strand cDNA can be made from
picogram amounts of total RNA.
• A potent RNase Inhibitor is included.
Applications
•First-strand cDNA synthesis.
•Production of cDNA for subsequent PCR or
real-time PCR.
•RT-PCR validation of gene expression data
obtained from microarray experiments.
•RT-PCR validation and quantification of gene
silencing by RNA interference.
FIG 1. The MMLV Reverse Transcriptase 1st-Strand
cDNA Synthesis Kit produces full-length cDNA
from mRNA longer than 15 kb. Total RNA isolated
from HeLa cells was reverse transcribed and the cDNA
was amplified as described in the text. A. Detection of
the 1.3 kb PCR amplicon from near the 5´ end of the
mRNA demonstrates full-length reverse transcription
of HERC1 mRNA. B. Agarose gel analysis of the PCR
reaction shows the 1.3 kb amplicon from the 5´-end
of the mRNA. Lane M, 100 bp DNA ladder; Lane 1, no
reverse transcriptase control reaction; Lane 2, PCR
product from cDNA synthesized by EPICENTRE’s MMLV
Reverse Transcriptase 1st-Strand cDNA Synthesis Kit.
MonsterScript™ 1st-Strand cDNA Synthesis Kit
The MonsterScript™ 1st-Strand cDNA
Synthesis Kit is optimized for generating
full-length first-strand cDNA from multiple
mRNA species in samples containing total
cellular or purified polyadenylated RNA. This kit
uses MonsterScript™ Reverse Transcriptase,
a reverse transcriptase that lacks RNase H
activity. The enzyme’s lack of RNase H activity
contributes to its ability to make longer cDNAs
and more complete libraries of first-strand cDNA
molecules.
Applications
•Lacks RNase H, enabling improved synthesis
of full-length cDNA even for long mRNA.
5'
•MonsterScript is thermostable, permitting
reverse transcription at temperatures >50°C,
which reduces RNA secondary structure and
improves priming specificity.
•MonsterScript™ RT PreMix contains
optimized concentrations of dNTPS, Mg+2
and Betaine for superior performance and
minimal pipetting steps.
•Betaine in the cDNA Synthesis PreMix
reduces pausing and stops during reverse
transcription.
•The kit includes both an oligo(dT)-containing
and a random nonamer primer.
•First-strand cDNA can be made from
picogram amounts of total RNA.
•A potent RNase Inhibitor is included.
8
• First-strand cDNA synthesis.
•Production of cDNA for subsequent PCR or
real-time PCR.
•RT-PCR validation of gene expression data
obtained from microarray experiments.
•RT-PCR validation and quantification of gene
silencing by RNA interference.
~ 15.2 kb HGNEFp532 mRNA
1A
AAAAA . . . -3'
i470407ms
Catalog No.
Conc.
Size
MonsterScript™ Reverse Transcriptase
MSTA5110
10 Reactions
MSTA5124
24 Reactions
1.3 kb
PCR
1B
—1.3 kb
FIG 1. MonsterScript™ Reverse Transcriptase
produces full-length cDNA from mRNA greater
than 15 kb. The ≈15.2 kb HGNEFp532 mRNA
was reverse transcribed from total HeLa RNA in a
standard MonsterScript reaction. Two microliters of
the reaction was used to PCR amplify a 1.3 kb region
within 68 bases of the 5´-end of the HGNEFp532
mRNA (1A). Agarose gel of the 1.3 kb amplicon from
the 5´-end of the mRNA demonstrates full-length
cDNA synthesis (1B).
[email protected] • www.EpiBio.com
Properties of Exonucleases and Endonucleases: Active on both DNA and RNA
Enzyme
Terminator™
5´-PhosphateDependent
Exonuclease
Substrate
Activity
Products
Applications
Heat
Inactivate
ssDNA
or
ssRNA
5´→ 3´ exonuclease that
digests ssDNA or ssRNA with
5´-phosphorylated ends, but
not with 5´-hydroxylated ends.
dNMPs or NMPs
Removal of 5’-phosphorylated DNA or primers
or oligos. Enrichment of ssDNA or ssRNA
molecules lacking 5’-phosphate groups.
65°C for
10 min
Single-strand-specific
endonuclease.
dNMPs or NMPs
and oligos with
5´-phosphate and
3´-OH
Converting 5´ or 3´ overhangs to blunt ends.
Removing hairpins after cDNA synthesis.
Detecting SNPs by digesting single-base
mismatches. With exo III, for making nested
deletions.“S1-type” mapping of RNA.
No
Endonuclease that efficiently
digests DNA and RNA.
di-, tri-, and
tetra-nucleotides
Removal of DNA and RNA from protein preps.
Removal of host DNA from phage preps.
No
Nucleases
DNA and RNA Exonuclease
DNA and RNA Endonucleases
Mung Bean
Nuclease
OmniCleave™
Endonuclease
ssDNA
or
ssRNA
single- and
double-stranded
DNA and RNA
Terminator™ 5´-Phosphate-Dependent Exonuclease
Terminator Exonuclease is a 5´-to-3´, processive
exonuclease that degrades RNAs that have a
5´-monophosphate. RNAs with a 5-triphoaphate,
5´-cap structure such as found on most
eukaryotic mRNAs or a 5´-OH are resistant to
Terminator Exonuclease degradation. It will also
digest DNA with a 5´-monophosphate. It is not
inhibited by proteinaceous RNase inhibitors.
Applications
•Characterize the 5-terminii of RNA transcripts.
•Prepare mRNA-enriched samples from
eukaryotic or prokaryotic total RNA
preparations in 1 hour without the use of
Oligo(dT), resins or magnetic beads.
Catalog No.
Conc.
Size
Terminator™ 5´-Phosphate-Dependent
Exonuclease
TER51020
1 U/µl
20 U
*Patent Pending.
Terminator™
Exonuclease
1 Hour
Large
rRNA
mRNA
FIG 2. A 1-hour Terminator™ Exonuclease
reaction digests the large rRNAs in a eukaryotic
or prokaryotic total RNA sample, producing an
enriched-mRNA preparation.
FIG 3. Denaturing agarose gel analysis of E. coli
total RNA before (-) and after (+) Terminator™
Exonuclease digestion. The Terminator Exonucleasetreated RNA was concentrated 10-fold.
FIG 4. Normal rat kidney (NRK) total RNA before (A) and after (B) Terminator™ Exonuclease treatment. The
Terminator Exonuclease-treated RNA was concentrated 10-fold.
www.EpiBio.com • [email protected]
9
Nucleases
Mung Bean Nuclease
Catalog No.
Conc.
Mung Bean Nuclease
M8202K
50 U/µl
M8205K
50 U/µl
This single-strand-specific nuclease has higher
specificity for single-stranded DNA and RNA
than S1 Nuclease. Unlike S1 Nuclease, Mung
Bean Nuclease will not cleave the intact strand
of nicked duplex DNA.
Size
2,000 U
5,000 U
Applications
•High resolution mapping of the termini and
exon structures of RNA transcripts (commonly
termed Berk-Sharp or S1-Mapping) using
either internal-labeled or end-labeled probes.
•Restriction site modification or removal by
digestion of single-stranded protruding ends.
•Removal of hairpin structures during cDNA
synthesis.
•Cleavage of single-basepair mismatches.
Digests all forms of DNA and RNA including
single-stranded and double-stranded linear,
circular, and supercoiled. OmniCleave
Endonuclease has the same substrate specificity
and yields the same products as Benzonase®,
an enzyme derived from Serratia marcescens.
Applications
OmniCleave™ Endonuclease
Catalog No.
Conc.
Size
OmniCleave™ Endonuclease
OC7810K
200 U/µl 10,000 U
OC7850K
200 U/µl 50,000 U
Provided with Dilution Buffer.
•Improves handling and yield of protein
preparations by reducing the viscosity of cell
lysates due to nucleic acids.
•Removes trace contamination by nucleic
acids in protein preparations.
•Removes host DNA from phage preparations.
Plasmid-Safe™ ATP-Dependent DNase
Catalog No.
Conc.
Size
Plasmid-Safe™ ATP-Dependent DNase
E3101K
10 U/µl
1,000 U
E3105K
10 U/µl
5,000 U
E3110K
10 U/µl 10,000 U
Plasmid-Safe™ ATP-Dependent DNase
selectively removes contaminating bacterial
chromosomal DNA from cosmid, BAC, fosmid,
and plasmid preparations. The enzyme will
processively degrade linear DNA from the
ends; closed circular DNA (i.e. a plasmid)
does not have free ends, and is therefore not
degraded. These properties make PlasmidSafe™ ATP-Dependent DNase ideal for BAC and
fosmid purification protocols such as shot-gun
sequencing and FISH where high purity DNA is
a must.
Applications
•Removal of contaminating bacterial
chromosomal DNA in large-scale plasmid,
cosmid, fosmid, BAC or vector preparations.
FIG 1. Plasmid-Safe™ ATPDependent DNase removes
contaminating genomic DNA from
plasmid preps. –, mixture of 3 µg of
digested bacterial chromosomal DNA
and 500 ng of uncut plasmid before
Plasmid-Safe™ DNase treatment;
+, mixture of chromosomal DNA and
plasmid DNA after Plasmid-Safe™
DNase treatment (incubated with
Plasmid-Safe™ DNase for 30 minutes
at 37°C); M, kb ladder.
Properties of DNA Endonucleases
DNA Endonucleases
Enzyme
10
Substrate
Activity
Products
Applications
Heat
Inactivate
Baseline-ZERO™
DNase
dsDNA
and
ssDNA
Digests dsDNA or ssDNA down to
mononucleotides
mononucleotides
Removing DNA from RNA
prep
75°C for
20 min
DNase I (bovine
pancreas)
E.C. 3.1.21.1
dsDNA
and
ssDNA
Activated by divalent cations. In presence of
Mg2+, it cleaves each DNA strand randomly
and independently, preferentially adjacent to
pyrimidines. In presence of Mn2+, it cleaves both
strands simultaneously, generating fragments
with blunt ends or 1-2-base overhangs.
Oligos and dNMPs
with 5´-phosphate
and 3´-OH.
Removing DNA from RNA
preps. Random nicking of
dsDNA. DNase footprinting.
75°C for
20 min
Endonuclease IV
(E. coli)
dsDNA with
abasic site
Cleaves sugar-phosphate bond 5´ of an abasic
site.
dsDNA with singlenucleotide gaps.
The cleaved ssDNA
strand has a 3´-OH.
DNA repair and anti-tumor
drug research. Base Excision
Sequence Scanning of DNA
containing dUMPs.
N/A
T4 Endonuclease V
UV-irradiated
DNA with
thymine
dimers
First cleaves N-glycosidic bond 5´ of thymine
dimers, then cleaves sugar-phosphate bond 3´
of the abasic site.
Nicked dsDNA with
an abasic site at
the 3´-end of the
cut and thyminedimer bases at the
5´-end of the cut.
Research on repair of DNA
exposed to UV light.
N/A
Lambda
Terminase
dsDNA with
cos sites
Cleaves both strands at bacteriophage
lambda cos sites.
5´-ends with
overhangs 12
bases in length.
Rapid sizing or restriction
mapping of BAC, fosmid or
cosmid clones.
N/A
[email protected] • www.EpiBio.com
Baseline-ZERO™ DNase
Provides a true zero baseline for RNA RT-PCR or
microarray gene expression experiments.
Applications
•Removal of genomic DNA from RNA prior to
RT-PCR or preparation of target RNA or cDNA
for microarray analysis, esp. for exon arrays
or full coverage expression analysis
•Elimination of the DNA template following in
vitro RNA synthesis with T7, T3 or SP6 Phage
RNA Polymerases.
•Removal of ssDNA and dsDNA from viral RNA.
•Elimination of genomic DNA from RNA for
microinjection and transfection experiments.
•As a replacement of DNase I in applications
requiring the removal of all DNA contamination.
•Reverse transcription of RNA using a random
primer since any contaminating DNA would
also be a template for random-primed cDNA
synthesis.
Catalog No.
Conc.
Baseline-ZERO™ DNase
DB0711K
1 U/ul
DB0715K
1 U/ul
FIG. Demonstration of
the use of BaselineZERO™ DNase
to remove small
oligonucleotides during
DNase treatment. Lane
1, kilobase ladder; Lanes
2-5, 160 ng of EcoR
I-digested plasmid DNA
incubated for 15 minutes
at 37°C as follows: Lane
2, untreated; Lane 3,
incubated with DNase I:
Lane 4, with supplier A’s
hyper-active DNase; Lane
5, with Baseline-ZERO™
DNase. Only BaselineZERO™ DNase removes
the small residual oligos at
the bottom of the gel.
*Patent Pending
Size
1,000 MBU
5,000 MBU
*Special introductory prices
Nucleases
Digests double- and single-stranded DNA to
mononucleotides more effectively than the
commonly used bovine pancreatic DNase I.
Following treatment with Baseline-ZERO, even
the small DNA oligonucleotides that remain after
treatment with bovine pancreatic DNase I are
undetectable.
Oligos
1 2 3 4 5
RNase-Free DNase I
RNase-Free DNase I (bovine pancreas) is an
endonuclease useful in removing DNA that might
interfere with the characterization, manipulation,
or use of RNA, or for any application requiring
highly purified DNase I. It efficiently hydrolyzes
double- and single-stranded DNA to a mixture of
short oligo- and mononucleotides.
1
2
3
—DNA
•Labeling of DNA by nick translation, in
combination with Klenow or other DNA
polymerases.
• Treatment of RNA prior to RT-PCR.
•Characterization of DNA-protein interactions
by DNase I footprinting.
Catalog No.
Conc.
RNase-Free DNase I
D9902K
D9905K
-
Size
2,500 MBU
5,000 MBU
Supplied at a concentration of 1 U/µl.
—transcript
Applications
•Elimination of template DNA following in vitro
synthesis of RNA with T7, SP6, or T3 phage
RNA polymerase.
4
FIG 1. Complete DNA removal from in vitro
transcription reactions using RNase-Free DNase I. A
linearized DNA template was transcribed using T7 RNA
polymerase according to standard in vitro transcription
conditions. Lane 1, kb ladder; Lane 2, DNA control;
Lane 3, transcription mixture; Lane 4, transcription
mixture treated with 1 MBU of RNase-Free DNaseI for
15 minutes at 37°C.
Endonuclease IV, E. coli
Endonuclease IV, cloned from the E. coli nfo
gene, is a metalloenzyme that functions in
vivo to repair free radical damage in DNA. The
enzyme also has Class II abasic endonuclease
activity, which has utility in many areas of DNA
damage and repair research. It is also useful in
the study of the effects of anti-tumor drugs such
as bleomycin on nucleic acids in vivo.
Applications
Catalog No.
Conc.
Endonuclease IV, E. coli
E70100
2 U/µl
Size
100 U
•DNA repair research.
•Anti-tumor drug evaluation.
T4 Endonuclease V
T4 Endonuclease V has N-glycosylase and
apurinic/apyrimidinic lyase (AP lyase) activities.
Ultraviolet (UV) light produces covalent
photoproducts in DNA, the most prevalent being
a cis-syn cyclobutane pyrimidine dimer. T4
Endonuclease V locates and binds to pyrimidine
dimers in double-stranded DNA, then cleaves
the N-glycosylic bond of the 5´-pyrimidine of
the dimer (pyrimidine dimer DNA glycosylase)
www.EpiBio.com • [email protected]
and breaks the phosphodiester bond 3´ to the
resulting abasic site (3´ AP lyase).
Applications
•Study of UV damage to DNA and its repair,
including DNA damage in single cells.
Catalog No.
Conc.
T4 Endonuclease V
TE6605
20 U/µl
TE661K
20 U/µl
TE665K
20 U/µl
Size
500 U
1,000 U
5,000 U
•Detection of differential UV damage repair of
transcribed sequences.
•Detection of UV mutational hotspots.
11
Nucleases
Lambda Terminase
Catalog No.
Conc.
Lambda Terminase
LT4450
2 U/µl
LT44200
2 U/µl
Size
50 U
200 U
Includes 10X Reaction Buffer and 10 mM ATP Solution.
This endonuclease encoded by bacteriophage
lambda recognizes and cleaves DNA at cos
sites, generating 5´-overhangs of 12 bases in
length. Since the 12-base cos site sequence
is rare, the sizes of inserts in BAC, fosmid, or
cosmid clones can be rapidly determined. The
clones are linearized with lambda terminase and
separated by pulsed field gel electrophoresis.
Lambda Terminase can also be used for
chromosomal mapping and for generating
restriction maps of DNA cloned into BAC,
cosmid, or fosmid vectors.
•Specific cleavage of chromosomal DNA for
physical mapping.
Applications
•Rapid sizing of BAC, fosmid, or cosmid
clones.
•Generation of restriction maps of BAC,
cosmid, or fosmid clone inserts.
•Linearization of cos site-containing clones for
in vitro packaging.
FIG 1. Digestion of BAC clones with Lambda
Terminase gives one band while digestion with Not
I often gives multiple bands. DNA from six randomly
chosen BAC clones were digested with Not I (Panel
1) or Lambda Terminase (Panel 2) Lane M: Lambda
Ladder PFG marker, Lane 1-6: BAC clones, Lane BT:
BAC-Tracker™ Supercoiled Ladder.
Properties of DNA Exonucleases
DNA Exonucleases
Enzyme
Exonuclease I
Substrate
Activity
Products
Applications
dNMPs
Removal of ssDNA and
oligonucleotides.
80°C for
15 min
Used with S1 Nuclease or
Mung Bean Nuclease to make
nested deletions. Preparation
of ssDNA templates for
sequencing. Site-directed
mutagenesis. Preparation of
labeled strand-specific probes.
70°C for
20 min
Removal of primers and
single-stranded oligos.
95ºC for
10 min
ssDNA
3´→ 5´ exonuclease
Exonuclease III
(E. coli)
dsDNA
3´→ 5´ exonuclease that digests duplex DNA from
the 3´-end of a nick or a blunt or 3´-recessed end; not
active on thionucleotides. Exo III also has RNase H,
3´-DNA phosphatase and apurinic DNA endonuclease
activities.
Exonuclease VII
ssDNA
Exonuclease that digests in both 5´→ 3´ and 3´→ 5´
directions.
Lambda
Exonuclease
dsDNA
5´→ 3´ exonuclease that digests dsDNA from 5´phosphorylated blunt or recessed ends. It has low
activity on 5´-hydroxylated ends and is not active on
nicked DNA.
RecBCD
Nuclease
(E. coli)
dsDNA
and
ssDNA
An ATP- and Mg2+- dependent exonuclease that digests
linear DNA in both 5´→ 3´ and 3´→ 5´ directions. Not
active on nicked or closed-circular dsDNA.
dNMPs
Removal of linear DNA from
circular DNA.
Rec J
Exonuclease
ssDNA
5´→ 3´ exonuclease that digests ssDNA with a 5´phosphate or a 5´-OH.
dNMPs
Removal of primers and
ssDNA from dsDNA.
T5
Exonuclease
dsDNA
and
ssDNA
5´→ 3´ exonuclease that also has single-strandspecific endonuclease activity in presence of
1-10 mM Mg2+ ions. At <1 mM Mg2+, the 5´→ 3´
exonuclease can digest from a nick in closed-circular
dsDNA without digesting the opposite strand.
dNMPs and ssDNA
on the opposite
strand. Partial
digestion produces
dsDNA having 5´
extensions of ssDNA.
dNMPs
dNMPs and ssDNA
on the the opposite
strand. Partial
Preparation of ssDNA
digestion produces templates for sequencing.
dsDNA having 3´
extensions of ssDNA.
dNMPs. Circular
ssDNA is obtained
from closed-circular
dsDNA in presence
of <1 mM Mg2+.
Heat
Inactivate
Plasmid mutagenesis.
Oligonucleotide site-directed
mutagenesis. Removing linear
DNA from plasmid preps.
Preparation of circular ssDNA.
75°C for
10 min
N/A
65°C for
20 min
N/A
Exonuclease I, E. coli
Catalog No.
Conc.
Exonuclease I, E. coli
X40501K
20 U/µl
X40505K
20 U/µl
12
Size
1,000 U
5,000 U
Exonuclease I digests single-stranded DNA
(ssDNA) in a 3´→ 5´ direction, but does not
digest double-stranded DNA (dsDNA). Although
it requires the presence of magnesium and a
free 3´-hydroxyl terminus for activity, it is active
under a wide variety of buffer conditions and
can be added directly into most reaction mixes.
It can be heat-inactivated by incubation at 80°C
for 15 minutes.
Applications
•Removal of residual ssDNA, including oligos,
from reaction mixes.
•Removal of ssDNA from nucleic acid mixtures.
[email protected] • www.EpiBio.com
Exonuclease III, E. coli
•Production of intermediates for site-directed
mutagenesis protocols.
•Production of strand-specific radiolabeled
probes.
Catalog No.
Conc.
Exonuclease III, E. coli
EX4405K
200 U/µl
EX4425K
200 U/µl
Size
5,000 U
25,000 U
Nucleases
Applications
Exonuclease III digests duplex DNA in a 3´→ 5´
direction from a nick or a blunt or 3´-recessed
end, producing stretches of single-stranded DNA
on the opposite strand.
Exonuclease VII
Exonuclease VII has high enzymatic specificity
for single-stranded DNA and exhibits both
5´→ 3´ and 3´→ 5´ exonuclease activities.
It is especially useful for rapid removal of
single-stranded oligonucleotide primers from a
completed PCR reaction when different primers
are required for subsequent PCR reactions.
Exonuclease VII digestion of single-stranded
DNA occurs in the absence of magnesium.
Applications
•Removal of single-stranded oligonucleotide
primers after PCR.
Catalog No.
Conc.
Exonuclease VII
EN510100
10 U/µl
EN510250
10 U/µl
Size
100 U
250 U
•Minimize the effect of primers left over from
previous PCR reactions.
Lambda Exonuclease
Applications
This highly processive 5´→ 3´
exodeoxyribonuclease selectively digests the
phosphorylated strand of double-stranded
DNA. The preferred substrate is blunt-ended,
5´-phosphorylated double-stranded-DNA. The
enzyme has reduced activity against nicked DNA
and against single-stranded DNA and gapped
DNA.
•SSCP (single-strand conformation
polymorphism) analysis of PCR product.
•Generate single-stranded DNA sequencing
template from PCR product.
Catalog No.
Conc.
Lambda Exonuclease
LE035H
500 U
LE032K
2,500 U
Size
10 U/µl
10 U/µl
Lambda Exonuclease
(5'-phosphate-specific 5'
5'OH
3' exodeoxyribonuclease)
OH
P
OH
OH
P
OH
P
PCR using one primer
with a 5'-phosphate
5'OH
OH
OH
P 5'OH
OH
OH
P
PCR product with strand-specific 5'-phosphate
Treatment with
Lambda Exonuclease
5'OH
5'OH
OH
OH
Single-stranded PCR product
dNMPs
FIG 1. Lambda Exonuclease selectively digests the strand of a PCR product produced using a PCR primer with
a 5´-phosphate. The resulting single-stranded PCR product can be used for SSCP analysis or sequencing.
RecBCD Nuclease, E. coli
This exonuclease from E. coli degrades singleand double-stranded DNA. Hydrolysis of the
DNA is bi-directional from both the 3´ and 5´
ends and processive, producing nucleoside
monophosphates. Magnesium is required for
the exonuclease activity, while calcium, nickel,
zinc, and copper inhibit exonuclease activity.
Calcium allows double-stranded DNA unwinding
(helicase activity) without hydrolysis.
www.EpiBio.com • [email protected]
Applications
•Removal of contaminating bacterial
chromosomal DNA in plasmid, fosmid,
cosmid, and BAC clone or vector preparation.
Catalog No.
Conc.
RecBCD Nuclease, E. coli
BCD0401K
-
Size
1,000 U
13
Nucleases
Rec J Exonuclease
Catalog No.
Conc.
Rec J Exonuclease
RJ411050
10 U/µl
RJ411250
10 U/µl
Size
50 U
250 U
Rec J Exonuclease, derived from E. coli,
catalyzes removal of deoxyribonucleoside
monophosphates from single-stranded DNA
in a 5´→ 3´ direction. Its activity is dependent
on Mg+2. Rec J Exonuclease can be heatinactivated by incubation at 65°C for 20
minutes.
Applications
This highly efficient 5´→ 3´ exonuclease for
either single-stranded or duplex DNA has
a tightly associated single-strand-specific
endonuclease activity when used in the
presence of 1-10 mM divalent magnesium ions.
This activity may be selectively suppressed by
using low concentrations of magnesium ions
(< 1 mM), allowing nicked, double-stranded
circular DNA to be “gapped” to a singlestranded circular species. In the absence of
divalent metal cofactors, T5 Exonuclease is
able to bind to DNA with a single-strand arm
adjacent to a duplex DNA region.
•Removes primers from completed PCR
reactions.
•Degrades single-stranded linear DNA in
dsDNA and plasmid preps.
T5 Exonuclease
Catalog No.
Conc.
T5 Exonuclease
T5E4111K
10 U/µl
Size
1,000 U
•Plasmid mutagenesis methods.
•Oligonucleotide site-directed mutagenesis.
•Generation of plasmid-sequencing templates.
•Removal of denatured DNA from alkalinebased plasmid purification procedures for
improved cloning procedures.
Our Exclusive RNA Exonuclease
RNase R
Catalog No.
RNase R
RNR07250
Applications
Conc.
-
Size
250 U
Ribonuclease R is a magnesium-dependent 3´→
5´ exoribonuclease that digests essentially all
linear RNAs but will not digest lariat or circular
RNA structures. Intron RNA can be isolated from
total RNA samples by digestion with RNase R.
After digestion, only lariat structures that are
produced during pre-mRNA splicing of intron
regions remain.
Applications
•Alternative splicing studies.
•Gene expression studies.
•Intron cDNA production.
•Intronic screening of cDNA libraries.
Ribonuclease R (RNase R)
Pre-mRNA
Exon 1
Intron
Exon 2
Intron
Exon 1
Exon 2
Intron
mRNA
Exon 1
Exon 2
Intron
Lariat Only Remains
i2220706
RNase R Digestion
FIG 1. How RNase R works.
14
[email protected] • www.EpiBio.com
Properties of RNA Endonucleases
Substrate
RNase A
ssRNA
Cleaves ssRNA 3´ of pyrimidine
residues.
ssRNA
Cleaves ssRNA between all
dinucleotide pairs.
RNase III, E. coli
dsRNA
Cleaves dsRNA in presence of Mg2+
to 12-15-bp dsRNA. Cleaves dsRNA
in presence of 20 mM Co2+ or Mn2+
to 18-25-bp dsRNA.
dsRNA with 5´phosphate and
2-base 3´-overhangs
with 3´-OH
Random cleavage of long dsRNA to short
dsRNA. RNA interference (RNAi).
RNase H, E. coli
RNA in
RNA:DNA
hybrid
Cleaves RNA in RNA:DNA hybrid
without affecting unhybridized RNA
or DNA.
oligoribonucleotides
with 5´-phosphate
and 3´-OH
Elimination of RNA prior to second strand
cDNA synthesis. Removal of poly(A) tails
from mRNA hybridized to oligo(dT).
Hybridase™
Thermostable
RNase H
RNA in
RNA:DNA
hybrid
Cleaves RNA in RNA:DNA hybrid
without affecting unhybridized RNA
or DNA.
oligoribonucleotides
with 5´-phosphate
and 3´-OH
High-stringency hybrid selection. Highstringency mapping of mRNA structure.
Transcription-based amplification methods.
No
RNA mapping and structure studies.
Removal of RNA from DNA preps.
N/A
Removal of all RNA from genomic and
cloned DNA preps.
No
RNase I, E. coli
Activity
Products
Heat
Inactivate
Enzyme
Applications
oligoribonucleotides
with 3´-cytidine or
3´-uridine residues
Removal of RNA from DNA preps. RNase
protection assays. RNA mapping and
structure studies.
N/A
NMPs with 5´-OH
and 2’,3´-cyclic
monophosphate
Removal of RNA from DNA preps. RNase
protection assays. Mismatch detection of
single basepairs in RNA:RNA or RNA:DNA
hybrids.
70°C for
15 min
RNase T1,
Aspergillis
oryzae
ssRNA
Cleaves ssRNA 3´ of GMPs.
oligoribonucleotides
with 3´-GMP residues
RiboShredder™
RNase Blend
ssRNA
Efficiently degrades all RNA.
NMPs
Nucleases
RNA Endonucleases
No
5´phosphate
Studies on RNA structure, RNA
processing and maturation.
65°C for
10 min
RNase A
RNase A is an endoribonuclease that cleaves
single-stranded RNA at the 3´-end of pyrimidine
residues, forming oligoribonucleotides having 3´terminal pyrimidine-3´-phosphates. Pyrimidine3´-monophosphates are also released by RNase
A cleavage of adjacent pyrimidine nucleotides.
Modified RNA containing pyrimidine-2’-fluorodNMPs, such as modified RNA made by in vitro
transcription using EPICENTRE’s DuraScribe®
T7 & SP6 Transcription Kits is completely
resistant to cleavage by RNase A.
Catalog No.
Conc.
Ribonuclease A
MRNA092
-
Applications
Size
2 ml @
5 mg/ml
•Removal of RNA from DNA preparations.
•Removal of unhybridized regions of RNA from
DNA-RNA or RNA-RNA hybrids.
RNase I, E. coli
RNase I degrades single-stranded RNA to
nucleoside 3´-monophosphates via 2’, 3´ cyclic
monophosphate intermediates by cleaving
between all dinucleotide pairs. This enzyme is
completely inactivated by heating at 70°C for 15
minutes, eliminating the requirement to remove the
enzyme prior to many subsequent procedures.
Applications
•Removal of RNA from DNA preparations.
•RNase protection assays to detect singlebasepair mismatches in RNA:RNA and
RNA:DNA hybrids.
Catalog No.
Conc.
RNase I, E. coli
N6901K
10 U/µl
N6905K
10 U/µl
Size
1,000 U
5,000 U
Provided with Dilution Buffer.
RNase III, E. coli
This endoribonuclease specifically digests
double-stranded RNA (dsRNA) to dsRNA
fragments that have 2-base, 3´-overhangs.
Complete digestion of dsRNA results in dsRNA
fragments of 12-15 bp.
www.EpiBio.com • [email protected]
Applications
•Digest long dsRNA to short dsRNA.
•RNA structure studies.
Catalog No.
Conc.
RNase III, E. coli
RN02950
1 U/µl
Size
50 U
• RNA processing and maturation studies.
15
Nucleases
RNase H, E. coli
Catalog No.
Conc.
RNase H, E. coli
R52250
10 U/µl
R0601K
10 U/µl
Size
250 U
1,000 U
This endonuclease specifically degrades the
RNA in an RNA:DNA hybrid, without affecting
DNA or unhybridized RNA. It will not degrade
double-stranded DNA or single-stranded DNA
or RNA. E. coli RNase H is inactivated at 55°C in
20 minutes.
Applications
•Elimination of RNA prior to second-strand
synthesis of cDNA.
•Removal of poly(A) tails from messenger RNA
hybridized to oligo(dT).
•Specific cleavage of mRNAs after
hybridization to oligonucleotide probes.
•Specific destruction of “hybrid-arrested”
mRNAs during in vitro translation.
•Diagnostic assays using NASBA™
transcription-based amplification methods.
•Diagnostic assays based on the Cycling Probe
Technology.
•Template–dependent probe technologies.
Hybridase™ Thermostable RNase H*
Catalog No.
Conc.
Size
Hybridase™ Thermostable RNase H
H39100
100 U
H39500
500 U
EPICENTRE’s patented Hybridase™
Thermostable RNase H specifically degrades
the RNA in a DNA:RNA hybrid, without affecting
DNA or unhybridized RNA. It has optimal activity
above 65°C and can be used at temperatures
up to 95°C. The thermostability of the enzyme
permits it to be used at temperatures that give
the highest hybridization stringency for specific
DNA:RNA heteroduplexes, maximizing sensitivity
and selectivity while minimizing background due
to nonspecific hybridization.
Applications
This endoribonuclease specifically cuts RNA
or deaminated RNA at the 3´-end of guanosine
residues and adjacent nucleotides through a 2’,
3´-cyclic phosphate intermediate mechanism.
Applications
•High stringency hybrid selection.
•Diagnostic assay of specific target DNA
sequences by isothermal probe amplification.
•Transcription-based amplification methods
(e.g., NASBA™).
•High stringency mapping of mRNA structure.
•Applications that require specific hydrolysis of
the RNA in a DNA:RNA hybrid.
*Hybridase™ Thermostable RNase H is covered by U.S.
Patent Nos. 5,268,289; 5,459,055; and 5,500,370 assigned
to EPICENTRE. This product is accompanied by a limited
non-exclusive license for the purchaser to use the purchased
product solely for life science research. Contact EPICENTRE for
information on licenses to other uses.
RNase T1, Aspergillus oryzae
Catalog No.
Conc.
Size
RNase T1, Aspergillus oryzae
NT09100K
100,000 U
NT09500K
500,000 U
•RNA mapping and structure studies.
•Removal of RNA from DNA preparations.
•RNA protection assays.
RiboShredder™ RNase Blend
Catalog No.
Conc.
Size
RiboShredder™ RNase Blend
RS12100
100 U
RS12500
500 U
RiboShredder™ is a cocktail of potent RNases
that completely degrades unwanted RNA in
DNA purification procedures. This highly active
cocktail contains a proprietary optimized
blend of non-mammalian RNase enzymes.
RiboShredder RNase Blend degrades all RNA,
converting RNA to nucleoside monophosphates.
• Completely degrades RNA rapidly.
• DNase-free.
Applications
•Removal of RNA from genomic and cloned
DNA preparations.
16
FIG 1. Comparison of RNA-degrading capability of
RiboShredder™ RNase Blend versus commonlyused individual RNases or other RNase cocktails.
Nucleic acids from a standard alkaline lysis plasmid
preparation were treated as follows under standard
reaction conditions: Lane 1, RNase A; Lane 2, RNase I;
Lane 3, RNase T1, Lane 4, RiboShredder RNase Blend;
Lane 5, RNase A/RNase T1 cocktail; Lane 6, Untreated
Alkaline lysis plasmid prep; Lane 7, supercoiled DNA
ladder.
[email protected] • www.EpiBio.com
Enzyme
8-Oxoguanine-DNA
Excision Mix
(8-Oxo-G-DNA
glycosylase or Fpg
and Endonuclease IV)
Uracil-DNA Excision
Mix
(HK™-UNG and
Endonuclease IV)
HK™-UNG
Thermolabile UracilN-Glycosylase (UNG)
(UNG is also
called Uracil-DNA
Glycosylase or UDG)
Activity
Products
Applications
Heat
Inactivate
Cleaves sugar-phosphate
bond 5´ of 8-oxo-GMPs.
dsDNA with single-nucleotide gaps
where 8-oxo-G residues have been
removed, or ssDNA strands with
lengths equal to distances between
8-oxo-G residues.
Site-specific cleavage of DNA
at 8-oxo-G sites. Base Excision
Sequence Scanning of DNA
containing 8-oxo-GMPs.
N/A
Cleaves sugar-phosphate
bond 5´ of dUMPs.
dsDNA with single-nucleotide gaps
where dUMP residues have been
removed, or ssDNA strands with
lengths equal to distances between
dUMP residues.
Fragmentation of dUMPcontaining DNA. Site-specific
cleavage of DNA at dUMP
sites. Base Excision Sequence
Scanning of DNA containing
dUMP residues.
65°C for
10 min
Uracil base and abasic DNA. Abasic
sites can subsequently be cleaved by
AP lyases, such as Endonuclease IV,
or by treatment with heat or alkaline
bases.
Fragmentation of dUMPcontaining DNA. Site-specific
cleavage of DNA at dUMP
sites. Base Excision Sequence
Scanning of DNA containing
dUMP residues.
65°C for
10 min
Substrate
dsDNA
or ssDNA
containing
8-oxo-GMP
dsDNA
or ssDNA
containing
dUMP
dsDNA
or ssDNA
containing
dUMP
Removes uracil base
from dUMP residues in
DNA.
Nucleases
Properties of DNA Glycosylases and Excision Mixes
Base-Specific DNA Excision Mixes and DNA Glycosylases
8-Oxoguanine-DNA Excision Mix
8-Oxoguanine-DNA Excision Mix is a blend of
enzymes that allows site-specific or random
cleavage of DNA at oxidized guanine residues.
The positions of the oxidized guanine residues
in a DNA sequence can be mapped by sizing
cleavage fragments on a sequencing-type gel
from a fixed priming site, yielding data similar
to a G-lane dideoxy-sequencing reaction.
The enzyme mix, first depurinates oxidized
G-residues, then cleaves the deoxyribose
phosphate backbone at apurinic sites,
generating a “polished” 3´-hydroxyl end and
releasing a DNA fragment with an abasic 5´phosphorylated end. The minimum oligomer size
that will serve as a substrate for excision by the
8-Oxoguanine-DNA Excision Mix is 6 base pairs.
The resulting DNA fragments can be analyzed
by denaturing agarose gel or polyacrylamide gel
electrophoresis.
Catalog No.
Conc.
Size
8-Oxoguanine-DNA Excision Mix
OG51100
- 100 Reactions
Contents: 8-Oxoguanine-DNA Excision Enzyme Mix, Guanine
Oxidation Reagent, and 10X 8-OxoG-DNA Excision Reaction
Buffer.
Applications
•Mapping of G residues in any DNA.
•DNA repair studies.
Uracil-DNA Excision Mix
Uracil-DNA Excision Mix is a blend of enzymes
that cleave DNA at positions where uracil is
present in place of thymine. The Uracil-DNA
Excision Mix is useful for specific or random
cleavage of DNA or for DNA repair studies,
allowing mapping of uracil residues in any
DNA. Uracil-DNA glycosylase in the Excision
Mix removes uracil bases from DNA, creating
a single base gap and leaving the deoxyribose
phosphate backbone intact. Endonuclease IV in
the Excision Mix then cleaves the DNA at each
abasic site, leaving a 3´-hydroxyl end and an
abasic 5´-phosphorylated end. The minimum
oligomer size that will serve as a substrate is 6
base pairs. Uracil-DNA Excision Mix digestion
products can be analyzed by denaturing agarose
gel electrophoresis or denaturing polyacrylamide
gel electrophoresis.
Catalog No.
Conc.
Size
Uracil-DNA Excision Mix
UEM04100
- 100 Reactions
Contents: Uracil-DNA Excision Enzyme Mix and 10X Uracil
Excision Enzyme Buffer.
Applications
•Mapping of uracil-containing residues in any
DNA.
• Mapping CpG islands.
• DNA repair studies.
HK™-UNG Thermolabile Uracil N-Glycosylase
This Uracil N-Glycosylase (also known as uracilDNA glycosylase) hydrolyzes the N-glycosidic
bond between the deoxyribose sugar and uracil
in DNA containing deoxyuridine in place of
thymidine. HK-UNG is active on both single- and
double-stranded DNA that contains uracil, but
has no activity on RNA or 2’-deoxyuridine-5´monophosphate.
www.EpiBio.com • [email protected]
Fully active at 50°C; inactivated by a 10-minute
incubation at 65°C.
Applications
•Repair studies of abasic sites in doublestranded DNA.
Catalog No.
Conc.
Size
HK™-UNG Thermolabile Uracil N-Glycosylase
HU59100
1 U/µl 100 U
HU5901K
1 U/µl 1,000 U
Provided with Dilution Buffer.
17
Ligases
Properties of Ligases
Base-Specific DNA Excision Mixes and DNA Glycosylases
Name of Ligase
Ligation Temp
Cofactor
Type of Ends
Ligated
Fast-Link™ DNA Ligase
Ligation Template Required to Ligate
Blunt
Cohesive
Heat
Inactivate
Primary
Application
5-15’ @ 20°C
ATP
NO*
YES
YES
Rapid Cloning
10’ @ 65°C
T4 DNA Ligase
4°C - 25°C
ATP
NO*
YES
YES
Cloning
15’ @ 65°C
E. coli DNA Ligase
5°C - 20°C
NAD
YES; DNA Only
Weak Activity
YES
Make long cDNA
20’ @ 65°C
Ampligase
DNA Ligase
20°C - 95°C
NAD
YES; DNA Only
NO
YES
TemplateDependent Ligation
NO; Half-life
1 hr @ 95°C
CircLigase™ ssDNA Ligase
20°C - 65°C
ATP
NO
Ligates ssDNA
N/A
Make ssDNA Circles
5´ @ 100°C
Ligate RNA to DNA
Make chimeric
DNA and RNA
15’ @ 65°C
®
T4 RNA Ligase
37°C
ATP
NO
Ligates ssDNA
*These enzymes ligate blunt ends of dsDNA, but ligation is more efficient on a ligation template, which can be DNA or RNA.
Fast-Link™ DNA Ligation Kit
Catalog No.
Conc.
Size
Fast-Link™ DNA Ligation Kit
LK11025
25 Ligations
LK0750H
50 Ligations
Contents: Fast-Link™ DNA Ligase, Fast-Link™ 10X Ligation
Buffer, 10 mM ATP
*For a full line of cloning products, please visit
www.EpiBio.com/cloning.asp.
This kit uses a high-quality ligase, called
Fast-Link™ DNA Ligase, that was cloned at
EPICENTRE and then formulated to provide
extremely rapid high-efficiency DNA ligation.
Cohesive-end ligations can be performed in 5
minutes at room temperature. It can be used for
routine and high-throughput DNA cloning.
• Cohesive-end ligations in 5 minutes at room
temperature.
•Blunt-end ligations in 15 minutes at room
temperature
•Ligation of PCR product with A-overhangs in
1 hour at 16°C.
•Desalting of ligation products is not needed
prior to transformation.
•High efficiency in-gel ligation.
T4 DNA Ligase is a commonly used ATPdependent ligase for DNA cloning. It covalently
joins double-stranded DNA molecules having
5´-phosphorylated and 3´-hydroxylated blunt or
compatible cohesive ends produced by restriction
enzyme digestion or other enzymatic processes.
T4 DNA Ligase has no activity on single-stranded
nucleic acids. Following a ligation reaction, T4
DNA Ligase may be inactivated by incubation
at 65°C for 10 minutes. EPICENTRE’s T4 DNA
Ligase is the highest quality T4 DNA Ligase
commercially available.
This NAD+-dependent enzyme catalyzes the
formation of phosphodiester bonds between
complementary 3´-hydroxyl and 5´-phosphoryl
termini of double-stranded DNA. The enzyme
works best with cohesive dsDNA ends and is also
active on nicked DNA. Blunt ends can be ligated
in the presence of condensing reagents such as
polyethylene glycol or Ficoll. It is not effective for
formation of DNA-RNA or RNA-RNA hybrids.
Applications
T4 RNA Ligase catalyzes the formation of a
phosphodiester bond between a 5´-phosphorylterminated nucleic acid donor and a 3´-hydroxyl
nucleic acid. The enzyme is active RNA, DNA,
oligoribo and oligodeoxyribonucleotides, and
nucleotide derivatives.
Applications
Applications
•TA cloning.
•PCR blunt-end cloning.
•Genomic DNA cloning and subcloning.
•BAC library construction.
•cDNA cloning.
•Linker ligation.
T4 DNA Ligase, Cloned
Catalog No.
Conc.
T4 DNA Ligase, Cloned
L0805H
2 U/µl
L0810H
2 U/µl
LH805H
10 U/µl
LH810H
10 U/µl
Size
500 U
1,000 U
500 U
1,000 U
Includes 10X Reaction Buffer and a separate 25 mM ATP
Solution.
*For a full line of cloning products, please visit www.EpiBio.
com/cloning.asp.
Applications
•Ligation of blunt or cohesive-ended DNA
fragments.
•Repair of nicks in double-stranded nucleic acids.
E. coli DNA Ligase
Catalog No.
Conc.
E. coli DNA Ligase
DL04082H
10U/µl
Size
200 U
•Molecular cloning of dsDNA with cohesive
ends.
•Blunt-end ligation in presence of 10-15%
PEG and high concentrations of monovalent
cations.
•cDNA cloning of products from second strand
cDNA synthesis experiments.
T4 RNA Ligase
Catalog No.
T4 RNA Ligase
LR5010
LR5025
Conc.
5 U/µl
5 U/µl
Size
1,000 U
2,500 U
Includes 10X Reaction Buffer and a 10 mM ATP Solution.
•mRNA tagging to map and sequence 5´termini or as a step in cDNA synthesis (RACE).
•3´-End labeling of RNA species.
•Ligation of RNA and DNA species to form
circles, extended oligonucleotides, or RNADNA-containing oligonucleotides.
•Modifications or mutagenesis of RNA species.
18
[email protected] • www.EpiBio.com
Ampligase® Thermostable DNA Ligase
oligonucleotide multimers when nucleotide
repeats are present in a DNA template.
•Simultaneous mutagenesis of multiple sites:
Ampligase DNA Ligase can introduce single
or multiple point mutations at specific sites
by ordered ligation of PCR-amplified DNA
fragments that have had point mutations
introduced via mutant primers.
•Other ligation-based detection methods.
Ampligase® Enzyme and Buffer
A0102K
100 U/µl
2,500 U
A32250
5 U/µl
250 U
A3202K
5 U/µl
2,500 U
Ampligase® DNA Ligase
A0110K
100 U/µl
A0125K
100 U/µl
A3210K
5 U/µl
A3225K
5 U/µl
High specificity and stringency permits sensitive
detection of SNPs.
10,000 U
25,000 U
10,000 U
25,000 U
One unit of Ampligase™ is equal to as many as 15 units of
other thermostable DNA ligases. Please compare competitive
unit definitions. Supplied as enzyme only; Reaction Buffer
is not included.
Applications
•Repeat Expansion
Detection (RED): RED
is a ligation-based
method of genetic
screening that
detects DNA regions
comprised of multiple
nucleotide repeats.
Ampligase DNA Ligase
is used in a two-step
thermal cycling
reaction that generates
One unit of Ampligase™ is equal to as many as 15 units of
other thermostable DNA ligases. Please compare competitive
unit definitions. Contains Ampligase® DNA Ligase,
Ampligase® 10X Reaction Buffer, and Ligation Control DNA.
One unit of Ampligase™ is equal to as many as 15 units of
other thermostable DNA ligases. Please compare competitive
unit definitions. 25 µl of Ampligase® 10X Reaction Buffer is
supplied with each 50 units of Ampligase® DNA Ligase.
High thermostability allows ligation using highstringency hybridization conditions.
•Ligation Amplification
(Ligase Chain Reaction,
LCR): Ligation
Amplification can
distinguish between
DNA sequences that
differ by as little as
a single base-pair
and is a useful tool
for detection of
single nucleotide
polymorphisms (SNPs).
Catalog No.
Conc.
Size
Ampligase® DNA Ligase Kit
A8101
5 U/µl
1,000 U
A30201
5 U/µl
5,000 U
Ligases
Derived from a thermophilic bacterium, stable
and active at much higher temperatures
than conventional DNA ligases, this enzyme
catalyzes NAD-dependent ligation of adjacent
3´-hydroxylated and 5´-phosphorylated termini
in duplex DNA structures that are stable at high
temperatures. Its half-life is 48 hours at 65°C
and greater than 1 hour at 95°C. It has been
shown to be active for at least 500 thermal
cycles (94°C/80°C) or 16 hours of cycling, which
permits extremely high hybridization stringency
and ligation specificity. No detectable activity
in ligating blunt-ended DNA, RNA or RNA:DNA
hybrids.
X
X
X
Ampligase® 10X Reaction Buffer
A1905B
5 ml
Ampligase® 1X Storage Buffer
A3201S
-
1 ml
X
FIG 1. Schematic of mutation discovery and
screening using ligation amplification. The
existence of a point mutation at the site of ligation
interferes with oligonucleotide ligation, resulting in no
ligation product. The lack of an amplification product
indicates the presence of a point mutation at the
ligation site. Oligos can also be designed so ligation
occurs in the presence of the mutant template.
CircLigase™ ssDNA Ligase
This thermostable ATP-dependent ligase
catalyzes intramolecular ligation (i.e.,
circularization) of single-stranded DNA (ssDNA)
templates having a 5´-phosphate and a 3´hydroxyl group and ligates ends of ssDNA in
the absence of a complementary sequence. It
is therefore useful for making circular ssDNA
molecules from linear ssDNA. Circular ssDNA
molecules can be used as substrates for rolling
circle replication or rolling circle transcription.
Efficient single-stranded DNA ligase activity.
Circularizes single-stranded DNA of >30 bases.
Standard reaction conditions produce no
detectable single-stranded DNA concatamers or
concatameric DNA circles.
Applications
•Production of single-stranded DNA templates
for rolling circle replication or rolling circle
transcription experiments.
www.EpiBio.com • [email protected]
•Production of single-stranded DNA templates
for RNA polymerase and RNA polymerase
inhibitor assays.
FIG 1. CircLigase™
ssDNA Ligase
converts linear
ssDNA to circular
ssDNA. A 71-base
ssDNA oligo
was converted
to a circular
DNA form in a
reaction containing
CircLigase™ ssDNA
Ligase and ATP.
Lane M, DNA markers. Lane 1, 71-base ssDNA. Lane
2, circularization proceeds through an adenylated
intermediate. Lane 3, the closed circular nature of
the reaction product was confirmed by treating the
reaction with exonuclease I, which specifically digests
linear DNA.
Catalog No.
Conc.
Size
CircLigase™ ssDNA Ligase
CL4111K
1,000 U
CL4115K
5,000 U
Contents: CircLigase™ ssDNA Ligase, CircLigase™ 10X
Reaction Buffer, ATP, 50 mM MnCl2 CircLigase™ Linear
ssDNA Control Substratem, Water
*Circligase™ ssDNA Ligase is covered by intellectual
property rights licensed to EPICENTRE. The purchase of this
product conveys to the buyer the non-transferable right to
use the purchased product and components of the product
in research conducted by the buyer. The buyer cannot sell or
otherwise transfer this product or its components to a third
party and in particular, no rights are conveyed to the buyer
to use the product or its components for commercial use
purpose other than for research to gain information that is
used by the buyer.
19
Phosphatases and Kinase
Properties of EPICENTRE’S Phosphatases
Heat-Labile Alkaline Phosphatases
Phosphatase
APex™
Type of Ends Dephosphorylated
Reaction
Temp
Nucleotides
are Substrates
Active at pH
Heat
Inactivate
YES
YES
5.5-12
5´ @ 70°C
-
YES
~7-10
15’ @ 65°C
Reaction Time
5´Protruding
Blunt
5´Recessed
20° - 50°C
Optimal
@ 37°C
YES
YES
37°C
Varies with Substrate Amt
-
-
NTPhos™
APex™ Heat-Labile Alkaline Phosphatase
Catalog No.
Conc.
Size
APex™ Heat-Labile Alkaline Phosphatase
AP49010
1 Reaction/µl 10 Reactions
AP49050
1 Reaction/µl 50 Reactions
This is a new, innovative enzyme preparation
with improved performance over other alkaline
phosphatases. APex™ Phosphatase removes
the 5´-phosphate from all types of DNA ends,
including 5´ protruding, blunt, and 5´ recessed
ends, and from RNA ends. The enzyme is
irreversibly heat-inactivated by incubation at
70°C for 5 minutes.
active over a wide range of temperatures, pH,
salts and buffers.
•Active on blunt, 5´- and 3´-overhang
restricted DNA ends for compatibility with any
restriction enzyme or experimental design.
•One simple protocol for most applications.
Applications
•Fast, complete and irreversible heatinactivation for easy transition to next step;
no time-consuming substrate purification
with phenol:chloroform extraction.
•Dephosphorylation of DNA vectors prior to
cloning to prevent recircularization.
•Flexible and easy to use—add directly to
most RE buffers without supplementation;
•Dephosphorylation of DNA/RNA substrates for
other purposes.
The 5´-termini of many natural RNA molecules,
including most eukaryotic messenger RNAs,
viral RNAs, many small nuclear RNAs, and
heterogeneous nuclear RNAs, have a structure
called a “cap.” Tobacco Acid Pyrophosphatase
(TAP) hydrolyzes the phosphoric acid anhydride
bonds in the triphosphate bridge of the cap
structure, releasing the cap nucleoside and
generating a 5´-phosphorylated terminus on
the RNA molecule. The resulting “decapped”
5´-phosphorylated terminus may be ligated to a
3´-hydroxylated terminus using T4 RNA Ligase
or dephosphorylated using APex™ Heat-Labile
Alkaline Phosphatase for end labeling. Similarly,
TAP digests the triphosphate group at the 5´-end
of prokaryotic transcripts, generating an RNA
molecule with a 5´-phosphorylated terminus.
•5´ and 3´-end mapping of RNA.
Applications
FIG 1. How TAP works
•Preparation of 5´-nucleic acid termini for 5´end labeling with polynucleotide kinase.
Tobacco Acid Pyrophosphatase
•Ligation of oligoribonucleotides to TAP-treated
cellular RNA for construction of full-length
cDNA libraries.
•Mapping of transcription initiation sites for
eukaryotic and prokaryotic transcripts.
•Radiolabeling of RNA for use in sequencing or
as a hybridization probe.
GpppG
OH
Capped RNA
TAP Treatment
OH Decapped RNA
pG
RNA Ligase
•Preparation of templates for RACE (Rapid
Amplification of cDNA Ends).
pG
Ligated RNA
+
T4 Polynucleotide Kinase, Cloned
Catalog No.
Conc.
Size
T4 Polynucleotide Kinase, Cloned
P0505H
10 U/µl
500 U
P0501K
10 U/µl
1,500 U
Includes 10X Reaction Buffer without ATP. ATP is available
separately.
ATP Solution
R109AT
-
5 µmoles
Provided as 500 µl of a 10 mM solution, pH 7.0.
20
pG
T4 Polynucleotide Kinase (PNK) catalyzes the
transfer of the gamma-phosphate from ATP to
the 5´-hydroxyl of single- and double-stranded
DNA, RNA, and nucleoside 3´-monophosphates.
The enzyme also removes the 3´-phosphate
from 3´-phosphoryl polynucleotides,
deoxyribonucleoside 3´-monophosphates, and
deoxyribonucleoside 3´, 5´-diphosphates to form
a 3´-hydroxyl group.
Applications
pG
OH
+
additional ligation
products
• L abeling of 5´-termini of DNA and RNA for DNA
sequencing, blot-hybridization, or transcript
mapping.
•Phosphorylation of oligonucleotide linkers and
other DNA or RNA molecules prior to ligation,
or for use in ligation amplification with
Ampligase® Thermostable DNA Ligase.
•Preparation of labeled DNA or RNA molecular
weight markers for gel electrophoresis and
chromatography.
[email protected] • www.EpiBio.com
i490407tap2
Catalog No.
Conc.
Size
Tobacco Acid Pyrophosphatase (TAP)
T19050
50 U
T19250
250 U
EasyLyse™ Bacterial Protein Extraction Solution
• Rapid protein screening.
•Easy protein purification.
• Enzymatic studies.
• ELISA studies.
•Manual or robotic procedures.
Bacterial Lysis (%)
60
40
0
EasyLyse™
Supplier P
Contents: Lysis Buffer, Enzyme Mix, MgCl2 Solution.
c170308bnl
20
Catalog No.
Conc.
Size
EasyLyse™ Bacterial Protein Extraction Solution
500, 1-ml
Purifications
or
RP03750
48, 96-well
Microplates,
100 µl/well
FIG 2. E. coli Lactate
Dehydrogenase
(LDH) specific
activity expressed
as nmol/min/µg of
soluble cell protein.
EasyLyse™ Preserves Enzyme Activity
1.5
1.2
1.0
0.5
0.12
0.0
EasyLyse™
Supplier P
c180308bnl
Applications
80
Specific Activity of Soluble LDH (nmol/min/µg)
Gives higher yields of soluble protein.
FIG 1. Lysis of
E. coli aliquots
with soluble
protein quantities
expressed as a %
of total protein, as
determined by the
Coomassie Plus
Protein Assay.
EasyLyse™ Bacterial Lysis Efficiency
100
Lysozymes
This extraction solution is designed for lysing
bacterial cells for the isolation of proteins,
especially recombinant gene products
expressed in E. coli, without significant loss of
enzymatic activity. It contains a highly active
enzyme for cell lysis and a potent nuclease that
reduces extract viscosity by digesting all nucleic
acids in the sample. The EasyLyse Solution is
formulated as a homogeneous reagent for ease
of use in high-throughput applications without
sonication.
Table 1. Bacteria lysed with Ready-Lyse Lysozyme
Solution.
Gram-negative
Gram-positive
Escherichia coli
Salmonella typhimurium
Actinobacillus
pleuropneumoniae
Rhodobacter sphaeroides
Shewanella putrefaciens
Flavobacteria odoratum
Oerskovia xanthinolytica
Bacillus subtilis
Catalog No.
Conc.
Size
Ready-Lyse™ Lysozyme Solution
R1802M
2 X 106 U
R1810M
10 X 106 U
*Animal Product-Free Ready-Lyse™ Lysozyme Solution
is available.
**For a complete line of purification products, please visit
www.EpiBio.com.
Applications
•Lysis of Gram-negative or Gram-positive
bacteria for protein purification.
3 hr.
M
1 hr.
Ready-Lyse™ Lysozyme Solution is a nonmammalian, non-avian, recombinant lysozyme
preparation for the lysis of Gram-negative (such
as E. coli) and Gram-positive (such as Bacillus
sp.) bacteria. The specific activity of Ready-Lyse
Lysozyme is 200-fold higher than the specific
activity of egg white lysozyme. Also, unlike egg
white lysozyme, Ready-Lyse Lysozyme Solution
is stable at –20°C, eliminating the need to
prepare a fresh solution for each use. The use of
Ready-Lyse Lysozyme results in higher yields of
protein than can be obtained with standard egg
white lysozyme.
0 hr.
Ready-Lyse™ Lysozyme Solution for Protein Extraction
1
2 3 4
FIG 1. Use of Ready-Lyse™ Lysozyme Solution to
recover recombinant proteins. One ml of induced
cells from a recombinant E. coli clone was pelleted by
microcentrifugation before induction and at 1 and 3
hours after induction.
Ready-Lyse™ Lysozyme Solution for Nucleic Acid Extraction
Ready-Lyse™ Lysozyme Solution is a nonmammalian, non-avian, recombinant lysozyme
preparation for the lysis of many Gram-negative
bacteria such as Escherichia coli and Grampositive bacteria such as Bacillus subtilis. The
use of Ready-Lyse Lysozyme results in higher
yields of DNA and RNA than obtained with
standard egg white lysozyme.
that are used once and then discarded, as is
required with egg white lysozyme.
Due to its higher specific activity, less ReadyLyse is needed for lysis compared to egg white
lysozyme. This reduces loss of nucleic acid from
enzyme binding.
FIG 1. Lysis with Ready-Lyse™
Lysozyme increases yields of
nucleic acids. Approximately
50% of the DNA was lost due to
precipitation by egg white lysozyme
(EW), while Ready-Lyse Lysozyme
(RL) caused minimal precipitation
losses of DNA compared to control
(C) samples without lysozyme.
Supplied as a ready-to-use solution stable at
–20°C, so there is no need to prepare fresh
solution prior to each use or to freeze aliquots
www.EpiBio.com • [email protected]
Applications
•Lysis of Gram-negative and Gram-positive
bacteria for preparations of nucleic acids.
•In-well gel screening of recombinant DNA in
agarose gels.
Catalog No.
Conc.
Size
Ready-Lyse™ Lysozyme Solution
R1802M
2 X 106 U
R1810M
10 X 106 U
*Animal Product-Free Ready-Lyse™ Lysozyme Solution is
available.
**For a complete line of purification products, please visit
www.EpiBio.com.
Red Cell Lysis Solution
MRC0912H
1200 ml
Tissue & Cell Lysis Solution
MTC096H
600 ml
21
Other Protein products
RecA Protein, E. coli
Catalog No.
Conc.
RecA Protein, E. coli
RC44200
5 µg/µl
RC441MG
5 µg/µl
Size
200 µg
1 mg
This multi-functional DNA-binding protein
encoded by E.coli plays integral roles in both
homologous recombination and post-replicative
DNA repair mechanisms. In vitro, RecA Protein
helps promote homologous recombination
through a multiple-step ATP-dependent
pathway. Initially, the protein binds preferentially
to single-stranded DNA forming a nucleoprotein
filament. The filament complex binds to naked
duplex DNA and searches for regions of
homology. Once a region of homology is found,
strand displacement and exchange begins.
Applications
•Site-directed mutagenesis through
displacement loop structures.
•Targeted site-specific cleavage of small and
large DNA.
•Enrichment of target sequences from libraries
or other DNA pools.
•Visualization of DNA for electron microscopy.
•Cloning or other experiments involving
use of RecA Protein and a site-specific
oligonucleotide to block endonuclease
cleavage at the complementary site in a
target DNA molecule.
Single-Stranded DNA Binding Protein (SSB), E. coli
Catalog No.
Conc.
Size
Single-Stranded DNA Binding Protein (SSB)
SSB02200
2 mg/ml
200 µg
Single-Stranded DNA Binding binds singlestranded DNA with high specificity. In vivo, SSB
is involved in DNA replication, recombination,
and repair. In vitro, SSB enhances several
molecular biology applications by destabilizing
DNA secondary structure and increasing
the processivity of polymerases. E. coli SSB
is also required for in vitro transcription of
single-stranded DNA templates by MiniV™
RNA Polymerase, a transcriptionally-active
1,106-amino acid domain of the N4 virion RNA
polymerase.
Applications
•Transcription of ssDNA templates by MiniV™
RNAP.
•Targeting restriction endonuclease digestion
to any restriction enzyme site in cloned
single-stranded DNA.
•Enhance the specificity and yield of PCR
reactions.
•Improve DNA sequencing results through
regions with strong secondary structure.
•Site-directed mutagenesis when used in
conjunction with recA protein.
•Improve the processivity of DNA polymerases.
•DNA replication and recombination studies.
DNA Topoisomerase I, Vaccinia
Catalog No.
Conc.
Size
DNA Topoisomerase I, Vaccinia
VT710500
10 U/µl
500 U
VT7105K
10 U/µl
5,000 U
22
Topoisomerase I from vaccinia virus is a type
I eukaryotic topoisomerase that removes both
positive and negative superhelical turns (also
called right- and left-handed supercoils) from
covalently closed DNA. The product of the
reaction is a covalently closed, circular DNA with
fewer positive or negative superhelical turns.
DNA Topoisomerase I does not absolutely require
Mg2+ to function, although low concentrations of
magnesium ions may increase activity.
Applications
•Studying the effects of supercoiling on
transcription in vitro.
•Studying chromatin reconstitution in vitro.
•Determining the degree of supercoiling of
naturally occurring DNA.
•Detecting mutant plasmids that differ in
length by only one basepair.
•Increasing restriction endonuclease digestion
of resistant DNA substrates by “unwinding”
the DNA coils to expose restriction sites.
[email protected] • www.EpiBio.com
Tagetin™ RNA Polymerase Inhibitor
Applications
• RNA polymerase studies.
• Transcription studies.
Catalog No.
Conc.
Size
Tagetin™ RNA Polymerase Inhibitor
T9705H
20 U/µl
500 U
T9702K
20 U/µl
2,500 U
FIG 1. Tagetin Inhibitor
activity on E. coli RNA
Polymerase. Each gel
lane shows products of
a standard transcription
reaction using a
bacteriophage template,
1 U of E. coli RNA
Polymerase Holoenzyme,
and varying amounts of Tagetin Inhibitor. Lane 1, 100
U; Lane 2, 10 U; Lane 3, 1 U; Lane 4, 0.1 U; Lane
5, control without Tagetin Inhibitor. 50% inhibition of
transcription is seen at 1 U Tagetin Inhibitor per unit of
E. coli RNA Polymerase.
Enzyme and Protein Inhibitors
Tagetin™ RNA Polymerase Inhibitor is the only
compound known to potently and selectively
inhibit RNA polymerase III from a variety of
eukaryotic organisms including mammalian
cells, Saccharomyces cerevisiae, Drosophila
melanogaster, Bombyx mori, and Xenopus
laevis oocytes. It strongly inhibits Escherichia
coli RNA polymerase and plant chloroplast RNA
polymerase. Plant nuclear RNA polymerases
I, II, and III are much less sensitive to Tagetin
Inhibitor. Phage-encoded RNA polymerases such
as SP6 and T7 are also relatively insensitive.
With both eukaryotic and prokaryotic RNA
polymerases, the degree of inhibition is
template-dependent.
ScriptGuard™ RNase Inhibitor
ScriptGuard™ RNase Inhibitor is your best
defense against common RNases including
RNase A, RNase B, and RNase C. This
recombinant RNase inhibitor protein provides
reliable protection of your precious RNA samples
by binding strongly to RNases in a 1:1 ratio.
EPICENTRE’s ScriptGuard™ RNase Inhibitor is
free of unwanted contaminants that can plague
other commercially available preparations of
RNase inhibitors.
•A potent affinity for RNases (Ki>10-14 M)
ensures rapid inhibition even when trace
amounts of RNase are present.
•Free of detectable RNase or DNase activity
and mammalian DNA.
•Does not interfere with enzymes commonly
used to prepare or analyze RNA.
Catalog No.
Conc.
Size
ScriptGuard™ RNase Inhibitor
SRI6325
40 U/µl
2,500 U
SRI6310K
40 U/µl 10,000 U
•Less sensitive to oxidation than traditional
RNase inhibitors.
Applications
•Effectively inhibits the degradation of RNA by
eukaryotic RNases in a variety of applications,
including cDNA synthesis, RT-PCR and in vitro
transcription and translation.
Protein Transport Inhibitor Brefeldin A
Brefeldin A (BFA), a metabolite of the fungus
Eupenicillium brefeldianum, specifically
and reversibly blocks protein transport from
the endoplasmic reticulum (ER) to the Golgi
apparatus in many cell types and species.
These effects are generally accompanied by
distinct morphological changes, including the
apparent collapse of the Golgi stacks. The fast
and reversible redistribution of intracellular
membranes is accompanied by various specific
and reversible effects on cellular protein traffic,
including protein transport from the ER to the
Golgi, protein secretion, vesicular assembly,
antigen presentation, trans- and endocytosis,
and viral assembly and budding.
Applications
•Studying mechanisms of protein transport
and targeting.
•Inducing “retrograde transport” of proteins
normally resident in the Golgi into the ER.
•Blocking protein secretion in many cell types.
•Blocking antigen presentation by major
histocompatibility complex class I and class II
molecules.
•Reversibly arresting assembly and release of
viral particles.
Catalog No.
Brefeldin A
B901MG
B905MG
Conc.
-
Size
1 mg
5 mg (5 x 1 mg)
•Blocking the toxic effects of ricin, modeccin,
abrin, and Pseudomonas toxin in various cell
types.
•Mapping post-translational modifications
of cell-surface receptors and other
glycoproteins.
FIG 1. Treatment
of primary mouse
pituitary cells with
Brefeldin A. BFA
treatment results in
the redistribution of
Golgi membranes
into the endoplasmic
reticulum (+BFA) as seen when compared with an
untreated cell (-BFA). (Electron micrographs are
courtesy of J.A. Magner, Michael Reese Hospital,
University of Illinois, Chicago.)
Enzyme Storage Buffer
Composition
Applications
50% glycerol containing 50mM Tris-HCl (pH
7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and
0.1% Triton X-100.
• Enzyme storage or dilution.
www.EpiBio.com • [email protected]
Catalog No.
Conc.
Enzyme Storage Buffer
ESB4901
-
Size
1 ml
23
Specialty Enzymes for DNA and RNA Research
Contents
RNA Polymerases and Replicases
RNA Capping and Tailing Enzymes
DNA Polymerases
Reverse Transcriptase
Nucleases and Glycosylases
Ligases
Phosphatases and Kinase
Lysozymes
Other Protein products
Enzyme Inhibitors
page
page
page
page
page
page
page
page
page
page
3
4
6
8
9
18
20
21
22
23
EPICENTRE has the necessary infrastructure, qualified and trained personnel and Quality
Systems procedures that are required to manufacture molecular biology enzymes of high
purity and quality that will exceed the expectations of the customer.
EPICENTRE develops, manufactures and sells optimal enzyme systems and reagents for
life science research, diagnostics and pharmaceutical bioprocessing.
LARGE QUANTITIES & SPECIAL FORMULATIONS are available. Please call toll free (U.S.
only) 1-800-284-8474 to inquire about custom kits or enzyme formulations, highthroughput packaging, bulk orders, animal product-free reagents and manufacturing to
meet specific regulatory requirements.
EPICENTRE never stops introducing new enzymes for your research needs. Sign up with us
at www.EpiBio.com/reply_card.asp to keep updated.
USA: 800-284-8474
Tel: 608-258-3080
Fax: 608-258-3088
Technical Support
USA: 800-284-8474
[email protected]