brochure novagen enzimas pcr

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.

Service Clients

Tlphone 0800 699 620 (numro vert)

Fax 0800 348 630 (numro vert)

E-mail [email protected]

Service Technique

Freecall 0800 126 461 (numro vert)


Visit our website: www.merckbio.eu

Germany

Orders

Freecall 0800 6931 000

Tel 06196 564952

Freefax 0800 6236 100


Technical Support

Freecall 0800 100 3496



Republic of Ireland

Orders

Toll free 1800 409 445

Tel +44 115 943 0840

Fax +44 115 943 0951


Technical Support

Freecall 1800 409 445



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.

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