brochure novagen enzimas pcr
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7/30/2019 Brochure Novagen Enzimas PCR
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PCR Enzymesfeaturing high fidelityKOD DNA Polymerases
KOD DNA Polymerases
Have the highest fidelity of anycommercially available DNApolymerase
Give greater yields in shorter times
Amplify targets up to30 kbp (whenKOD XL DNA Polymerase is used)
Amplify difficult targets,including GC-rich sequences
Because onewrong note. .
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novagen.com/kod
We offer a complete selection of Novagen enzymes and
kits for PCR, featuring KOD DNA Polymerase. This unique
proofreading enzyme, isolated from the extreme thermo-phile Thermococcus kodakaraensis KOD1, possesses superior
processivity and fidelity that enables faster and more accurate
PCR amplification than can be achieved with conventional
enzymes, including Pfu DNA polymerase (Takagi 1997). KOD
DNA Polymerase is also available as a hot start version for
high specificity and increased read length (Mizuguchi 1999),
and as a blend (KOD XL DNA Polymerase) recommended for
very long templates (Nishioka 2001).
NovaTaq DNA Polymerase is a high-purity, recombinant
enzyme suitable for any application requiring premium qu
ityTaq DNA polymerase. For increased specificity and connience with standard PCR, we offer NovaTaq Hot Start DNA
Polymerase. NovaTaq Hot Start DNA Polymerase is a chem
cally modified form of the enzyme that becomes active wh
heated at 95C for 7 to 10 minutes. Please use the followin
Selection Guide to find the appropriate enzyme combinatio
for your application.
When fidelity matters
KOD DNA Polymerase
Features
Higher fidelity than PfuDNA polymeraseexcellent for
cloning
Greater yieldextension speed is 2X faster than TaqDNA
polymerase and 5X faster than PfuDNA polymerase
Higher processivitysequential nucleotide polymerization is
10- to 15-fold greater than Pfuand TliDNA polymerases No truncated amplification products
Amplifies plasmid and lambda DNA templates up to 6 kbp
Amplifies genomic DNA templates up to 2 kbp
KOD Hot Start DNA Polymerase
Features
Highest accuracy, yield, and processivity of commercially
available proofreading DNA polymerases
Amplifies genomic DNA templates up to 12 kbp
Amplifies plasmid and lambda DNA templates up to 21 kb
Successfully amplifies GC-rich sequences
Eliminates mispriming and primer-dimer formation Convenient ambient-temperature setup compatible with
automation
KOD Hot Start Buffer ensures optimal PCR performance o
a wide range of targets
Manufactured by and distributed by Merck, not available from Merck in Jap
PCR Enzyme Selection Guide
Enzyme PCR ProductSize kbpElongation Rate
bases/s Specificity FidelityGC-rich
Templates YieldPCR
Product Ends
KOD DNA Polymerase < 6 120 blunt
KOD Hot Start DNA Polymerase < 21 120 blunt
KOD XL DNA Polymerase < 30 120 Mixed
(blunt and 3-d
NovaTaq DNA Polymerase < 5 60 3-dA
NovaTaqHot Start DNA Polymerase < 5 60 3-dA
Satisfactory Good Excellent Best
. . . can ruin the whole performance!
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When amplifying DNA for cloning, high fidelity DNA polymer-
ases, such as KOD polymerase, are recommended. If the enzyme
is also fast and can generate high yields of full-length product,
fewer amplification cycles are required and the probability of
obtaining error-free clones is increased. Since the preliminary
KOD DNA Polymerase studies, significant work has been doneto optimize the PCR buffer and cycling parameters. A number
of independent studies have verified the extreme high fidelity of
KOD DNA Polymerase compared to other thermophilic polymer-
ases (Takagi 1997, Nishioka 2001, Rual 2004, Wu 2006). The data
below show the speed and yield of KOD and KOD Hot Start DNA
Polymerases compared to five other high fidelity thermophilic
DNA polymerases.
Cycling profilesAll 7 enzymes were tested in 4 different cycling protocols, which
encompass the manufacturers recommended cycling conditions
(Table 1).
Table 1. Cycling profiles
KOD yields more product in fewer cycles comparedto other PCR enzymes
M 1 2 3 4 M 5 6 7 M 1 2 3 4 M 5 6 7
M 1 2 3 4 M 5 6 7 M 1 2 3 4 M 5 6 7
Lane Sample
M PCR Markers
1 KOD Hot Start DNA2 KOD DNA Pol.3 Platinum Pfx DNA
4 Pfx50 DNA Pol.
5 Phusion Hot Start
6 PfuUltra II Fusion H
7 PrimeSTAR HS DNA
23 cycles 25 cycles
27 cycles 29 cycles(plus final extension)
PCR samples (5 l) were taken after 23, 25, 27, and 29 cycles and assayed on 1.4% agarose/TAE gels. Lanes indicate the enzyme used for the reaction. Cycling profiles are defined in Table1. (A) cycling profile A (Note: PicoGreen results indicate a yield increase with PrimeStar HSDNA Pol. from cycle 27 to cycle 29; the reduced band intensity is a gel artifact)
For cycles B, C, and D, please visit www.novagen.com/KOD/articleor request a copy of
inNovations 25.
Figure 1. Cycling profile A
Cycle Profile A Cycle Profile B Cycle Profile C Cycle Profile D
Initial denaturation 98C 30 s 94C 2 min 95C 2 min 95C 2 min
29 cycles
98C 10 s
55C 20 s
72C 30 s
94C 15 s
52C 20 s
68C 60 s
95C 20 s
55C 20 s
72C 30 s
95C 20 s
55C 10 s
70C 15 s
Final extension 72C 5 min 68C 5 min 72C 3 min N/A
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
19 21 23 25 27 29
l
l
ll
l
l
l
l
Cycle number
Yield
(Mg/25-Mlreaction)
Figure 2. Best yield for each high fidelity themophilic enzyme from any cycling profileKOD is Rapid
Yields were determined by PicoGreen
analysis after 19, 21, 23, 25, 27, and
29 cycles for all 4 cycling profiles
(Table 2). The best yield data for each
enzyme, from any cycling profile, was
graphed. The number of the cycling
profile that gave the best yields is listed
in parentheses after the enzyme name.
The green shaded area calls attentionto yields in cycles 19-25, which would
be preferable for cloning. The graph shows that KOD Hot
Start DNA Polymerase gives 12%392% higher yields at 25
cycles than the other enzymes tested.
In addition to a low mutation frequency, the fast extension
rate and high processivity of KOD result in higher yields
of full-length product in fewer reaction cycles. Combined,
these make KOD Hot Start DNA Polymerase the PCR enzyme
of choice for structural proteomics studies.
KOD Hot StartDNA Pol. (1)
KOD DNA Pol. (1)
Platinum PfxDNA Pol. (2)
Pfx50 DNA Pol. (2)
Phusion Hot StartDNA Pol. (3)
PfuUltra II Fusion HSDNA Polymerase (2)
PrimeSTAR HSDNA Pol. (1)
References
Mizuguchi, H. et al. 1999. J. Biochem. (Tokyo)126, 762.
Nishioka, M. et al. 2001. J. Biotech.88, 141.
Rual, J-F. et al. 2004. Genome Res.14, 2128.
Takagi, M. et al. 1997. Appl. Environ. Microbiol.63, 4509.
Wu, G. et al. 2006. J. Biotechnol. 124, 496.
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Manufactured by and distributed by Merck, not available fromMerck in Japan.
Product Size Cat No.
NovaTaqDNA
Polymerase
100 U 71003-3
500 U 71003-4
2500 U 71003-5
NovaTaqPCR
Master Mix
200 rxn 71007-3
NovaTaqHot Start DNA
Polymerase
250 U 71091-3
1250 U 71091-4
NovaTaqHot Start
Master Mix Kit
200 rxn 71676-3
1000 rxn 71676-4
For routine PCR
NovaTaq DNA Polymerase andMaster Mix Ultrapure recombinant enzyme for
dependable PCR amplification
Licensed for PCR
Master Mix is premixed 2X PCR components
for convenience and reproducibility
NovaTaqHot Start DNA Polymeraseand Master Mix Kit Heat-activatable, chemically modified
TaqDNA Polymerase
Licensed for PCR
Greater PCR specificity and yield
Improved low-copy target amplification
Ambient temperature setup compatible
with automation
Master Mix is premixed 2X PCR
components for convenience and
reproducibility
Theth
reeRs
ofPCR:Rapid,Robust,Reliable
For long and accurate PCR
KOD XL DNA PolymeraseFeatures
Ideal for amplification of large DNAfragments from purified DNA or crudesamples
Amplifies DNA templates up to 30 kbp Successfully amplifies GC-rich sequences Efficiently incorporates derivatized dNTPs
NewLower
Price
Lane DNA Polymerase
M PCR Markers
1 KOD Hot Start2 KOD3 Platinum Pfx
4 Pfx50
5 Phusion Hot Start
6 PfuUltra II Fusion HS
7 PrimeSTAR HS
M 1 2 3 4 M 5 6 7 M 1 2 3 4 M 5 6 7
27 cycles 29 cycles
(plus final extension)
1 2 3 4 M 5 6 7 M 1 2 3 4 M 5 6 7
KOD is RobustOD for 2-step PCRnger primers (generally 23 bases) can increase PCR specificity
d, due to higher annealing temperatures, can be used in time-
ving 2-step cycling profiles. KOD Hot Start DNA Polymerase
d the other 6 high fidelity enzymes were tested in a 2-stepotocol (initial denaturation 95C for 2 min, and 29 cycles of
5C for 20 s, 68C for 40 s). Figure 3 shows that not all en-
mes functioned well with this 2-step protocol. Other enzymes,
cluding the 2 KOD enzymes, generated high yields comparable
the 3-step protocols (Compare to Figure 1).
gure 3. PCR results from 7 high fidelity enzymessing a 2-step cycling profile
R samples were removed after 23, 25, 27, and 29 cycles of a 2-step protocol and
l were assayed on 1.4% agarose/TAE gels. Lanes indicate the enzyme used for thection.
KOD is ReliableOD application: screening cDNA librariesOD Hot Start DNA Polymerase was used in a 2-step cycling
ofile to screen clones from the T7Select Human cDNA
brary (Cat. No. 70637). Of the 50 clones screened, KOD suc-
ssfully amplified 49 inserts (Figure 4). Amplicons ranged in
ze from ~250-1800 bp.
gure 4. KOD Hot Start amplification of T7Selectuman cDNA Library
actions were cycled with an initial denaturation at 95C for 2 min, and 25 cycles of
C for 20 s, 68C for 25 s.
1 2 3 4 M2 5 6 7 8 M1 9 10 11 12 M2 13 14 15 16
17 18 19 20 M2 21 22 23 24 M1 25 26 27 28 M2 29 30 31 32
l
M1 33 34 35 M2 36 37 38 39 40 M1 41 42 43 44 M2 45 46 47 48
M1 49 5 0 M2
Lane Sample
M1 Perfect DNA Markers
M2 PCR Markers
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KOD DNA Polymerase
KOD Hot Start DNA Polymerase
KOD XL DNA Polymerase
As an active group dedicated to synthetic gene design and gene synthesis, we made
great efforts to improve the efficiency of synthetic gene construction. With the increas-
ing popularity of synthetic gene technology, the supply of an efficient and accurate
DNA polymerase becomes critical. We found that KOD DNA polymerase had out
performed some other polymerases(Taqand Pfu) in our Simplified GeneSynthesis method.Gang Wu, Ph.D.
Postdoctoral Fellow, Johns Hopkins University, Bloomberg School of Public Health
"KOD polymerase has dramatically changed how we think about PCR fidelity and using PCR for cloning.
With its high fidelity and processivity, we seldom see PCR-induced errors in any of ourcloned ORFs. The errors are now regularly associated with the oligos used as primers for PCR ratherthan the PCR product itself. In our large-scale cloning projects, we end up sequencing far fewer single
colony isolates to obtain wild type clones. We also can confidently use PCR to transfer target sequences
rather than relying on more labor-intensive and time consuming molecular cloning protocols."David E. Hill, Ph.D.Senior Research Scientist, Dana-Farber Cancer Institute
"In the Reetz lab here at the Max-Planck-Institute in Muelheim, Germany we
have been successfully using KOD Hot Start DNA Polymerase since the year 2004
for a variety of different applications. Besides routine PCR for cloning experi-
ments the focus of these applications was on mutagenesis by non-exponential
whole-plasmid amplification. For amplification of plasmids up to 7 kbpwe found KOD Hot Start to be especially robust and to show a highfidelity even under harsh reaction conditions. Based on our experience
using a range of different plasmids as templates we can strongly recommend the use of this enzyme."
Frank Schulz and Horst HbenreichGroup Prof. Dr. M. T. Reetz
Max-Planck-Institute for Coal Research
KOD out performs
Visit our KOD Web Page for more information: Applications Links to citations
www.novagen.com/KODProduct Size Cat. No. Price
250 U 71085-3
20 U 71086-5200 U 71086-3
1000 U 71086-4
250 U 71087-31250 U 71087-4
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"Recently I have been attempting site-
directed mutagenesis on a very large plasmid
(10.3 Kb) with PfuTurbo and PfuUltra DNAPolymerase. However I was having difficulty
in obtaining any positive clones and decided
to try KOD Hot Start DNA polymerase in my
reactions. With this enzyme I finally ob-
tained the mutations I wanted within
a short time-frame. The fidelity of KOD is
similar to that ofPfu, but its main strength
is its vastly increased elongation rate. As an
added bonus it turned out to be a lot cheaper in thelong run. In the future I would certainly
recommend KOD Hot Start as a place to start
for any project involving mutagenesis."
Gary Morley, Ph.D.Institute of Cell and Molecular Biosciences
University of Newcastle
Why is KOD thebest high fidelity
PCRenzyme?Look inside!
Direct sequencing of
~70,000 bases indicated a
misincorporation rate of
only 1 in 35,000 for KODHot Start DNA
Polymerase (Rual 2004)
Start withthe best KOD
France.
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E-mail [email protected]
Visit our website: www.merckbio.eu
For more information or to order Novagen products, contact Merck Chemicals Ltd.
220024-2007EU
KOD DNA Polymer
Prices and availability are subject to change. Copyright 2007 EMD Chemicals, an affiliate of Merck KGaA, Darmstadt, Germany. All Rights Reserved. Each product is sold with a limited war-ranty which is provided with each purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for humanuse. EMD Chemicals products may not be resold, modified for resale, or used to manufacture commercial products without written approval of EMD Chemicals. Novagen and T7Selectare registered trademarks of EMD Biosciences in the United States and in certain other jurisdictions. NovaTaq is a trademark of EMD Biosciences. PfuTurbo and PfuUltra are registeredtrademarks of Stratagene. Pfx50 is a trademark and Platinum is a registered trademark of Invitrogen Corp. Phusion is a trademark of Finnzymes Oy. PicoGreen is a registered trademarkof Molecular Probes, Inc. PrimeSTAR is a registered trademark of Takara Bio Inc.