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F0934K

Instruction manual KOD -Plus- 0902

KOD -PlusKOD-201

200 U

200 reactions

Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Quality testing[4]

Primer design[5]

Cloning of PCR products[6]

Protocol
1. Standard reaction setup
2. Cycling conditions[7]

Examples[8]

Troubleshooting[9]

References[10]

Related products

CAUSIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe
general laboratory precaution and utilize safety while using this kit.

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www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
KOD -Plus- is based on DNA polymerase from the hyperthermophilic Archaeon
Thermococcus kodakaraensis KOD11) 2). KOD -Plus- exhibits excellent high PCR fidelity
and efficiency. The enzyme solution of KOD -Plus- contains two types of anti-KOD
DNA polymerase antibodies that inhibit polymerase and 3’J5’ exonuclease activity, thus
allowing for Hot Start PCR3). KOD -Plus- generates blunt-end PCR products, due to
3’J5’ exonuclease (proof-reading) activity.

Features-Hot Start technology, using anti-KOD DNA polymerase antibodies, results in highlyefficient amplification (see Example 1).
-KOD -Plus- enables the following amplifications (maximum): 21 kb from lambda phage
DNA, 12 kb from human genomic DNA, and 7 kb from cDNA.
-KOD DNA polymerase has strong 3’J5’ exonuclease (proof-reading) activity. The PCRerror ratio of KOD -Plus- is approx. 80 times less than Taq DNA polymerase.

Table. 1

PCR fidelity comparison of each PCR enzyme.

KOD -PlusHigh fidelity PCR enzyme (A company)
Pfu based DNA polymerase
Taq DNA polymerase

Colony number
Total
Mutant10,61010
10,90068
6,520
76
10,560780

Mutation frequency
(%)
0.09
0.62
1.17
7.39

*PCR fidelity was based on the mutation frequency of PCR products using a positive-selection
base assay with the rpsL gene 4).

[ 2 ] Components

This reagent includes the following components for 200 reactions:
KOD -Plus- (1.0 U/μl) *10× Buffer for KOD -Plus25 mM MgSO4
2 mM dNTPs

200 μl × 1
1.0 ml × 1
1.0 ml × 1
1.0 ml × 1*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize
polymerase and 3’J5’ exonuclease activity.

[3]

Quality TestingJAPANTOYOBO CO., LTD.
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Quality check can be performed by amplifying the β-globin gene (3.6 Kb) and p53 gene(4.0 Kb).

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www.toyobo.co.jp/e/bio[4]Primer Design[ 5 ] Cloning ofPCR products-Primers should be 22-34 bases with a melting temperature (Tm) over 60°C. For
amplification of a long target, 25-34 bases with high Tm values (≥ 65°C) arerecommended. PCR primers should be designed according to the general guidelines.

-KOD-Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proofreading) activity. Therefore, the product can be cloned according to a blunt-end cloning
method.
-PCR products of KOD-Plus- should be purified prior to restriction enzyme treatments.
The 3’J5’ exonuclease activity of KOD DNA polymerase remains after the PCR cycles.

[ 6 ] Protocol

1. Standard reaction setup
The following procedure is designed for use with the components provided in this kit.
Before preparing mixture, all components should be completely thawed, except for theenzyme solution.
Component
10x Buffer for KOD -Plus2mM dNTPs*
25mM MgSO4
10pmol/μl Primer #1
10pmol/μl Primer #2Volume5 μl
5 μl
2 μl
1.5 μl
1.5 μlTemplate DNAX μl

PCR grade water
KOD-Plus- (1.0 U/μl)
Total reaction volumeY μl
1 μl
50 μl

Final Concentration
1x0.2 mM each
1.0 mM
0.3 μM
0.3 μM
Genomic DNA 10-200 ng/50 μl
Plasmid DNA 1-50 ng/50 μl
cDNA ≤ 100 ng （RNA equiv.)/50 μl

1.0 U / 50 μl* Do not use dNTPs from other kits or companies.
Notes:-For PCR reactions, thin-wall tubes are recommended. A total reaction volume of 50 μl
is also recommended.
-The addition of DMSO (final conc. 2-5%) might be effective for amplification ofGC-rich targets. Decreased PCR fidelity has been confirmed to not take place withDMSO.
-Contaminated RNA (used for cDNA) or genomic DNA inhibits the PCR reaction bychelating Mg2+. PCR should be performed using template DNA containing <100 ng
RNA component.
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2. Cycling conditions
The following cycling steps are recommended.
< 2-step cycle >Pre-denaturation:

94 °C , 2 min.

Denaturation:

94 °C, 15 sec.

Extension:

68 °C, 1 min./kb

25-35 cycles

Note: If the Tm value of the primer is under 73 °C, the 3-step cycle is recommended.

< 3-step cycle >Pre-denaturation:

94 °C, 2 min.

Denaturation:

94 °C, 15 sec.

Annealing:Tm-[5-10] oC*, 30 sec.

Extension:

68 °C, 1 min./kb

25-35 cycles*Tm value of the primer minus 5°C-10°C

Notes:-Extension time should be set to 1min per 1 kb of target length.

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www.toyobo.co.jp/e/bio[ 7 ] Examples
Example 1．Effect of Hot Start PCR on the generation of primer dimers.

M 1 2 3 4 56M
Template: Human genomic DNA
1,3,5: 50ng, 2,4,6: 100ng
Target: p53 gene 4kb
M: λ/HindⅢ Marker
1,2: KOD -Plus3,4: A company high fidelity enzyme
5,6: B company high fidelity enzyme

Primer dimer

Example 2. Effect of addition of DMSO for GC-rich targets.

M

123Template: Human genomic DNA
Target: TGF-β gene (GC%=70) 2kb
M: 1kb Ladder Markers
1: KOD -Plus-, 0% DMSO
2: KOD -Plus-, 2% DMSO
3: KOD -Plus-, 5% DMSOJAPANTOYOBO CO., LTD.
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www.toyobo.co.jp/e/bio[ 8 ] TroubleshootingSymptom

No PCR product/low yield

Cause

Solution

Cycling conditions are notsuitable.

Lower annealing temperature increments to a
maximum of Tm-10°C.
Increase the number of cycles by 2-5 cycles.
Increase the Mg concentration to 1.2-2 mM.
Add DMSO 2-5%.
<See Example 2>
Check the quality of primers.
Redesign primers.
Check the quality of template DNA. RNA inhibits
amplification.
Increase the amount of template DNA.
Decrease the number of cycles by 2-5 cycles.
Use step-down cycling.
Check the quality of primers.
Redesign primers.
Reduce the amount of template DNA.
Reduce MgSO4 to 0.8 mM.
Reduce enzyme to 0.5-0.8 U/50 μl.
Clone the PCR products according to general bluntend cloning guidelines.

Mg concentration is low
High GC content of target
sequence
Primer is not good.
Quality and/or quantity oftemplate DNA is not
sufficient.
Cycling condition is notsuitable.
Primer is not good.

Smearing/extra band

Poor TA cloning efficiency[ 9 ] References1)

Too much template DNA
Too much Mg
Too much enzyme
PCR products have bluntends.

Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M,and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)

2)

Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, JMol Biol., 306: 469-77 (2001)

3)

Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126:762-8 (1999)

4)

Fujii S, Akiyama M, Aoki K, Sugaya Y, Higuchi K, Hiraoka M, Miki Y, Saitoh N,
Yoshiyama K, Ihara K, Seki M, Ohtsubo E and Maki H, J. Mol. Biol., 289: 835-850(1999)JAPANTOYOBO CO., LTD.
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FOR RESEARCH USE ONLY.

5NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 10 ] Related products
Product name
TArget Clone -Plus10x A-attachment mix
Ligation high Ver.2Package
10 reactions
25 reactions
750 μl(100 reactions)

Code No.
TAK-201
TAK-301
LGK-201

TArget Clone -Plus- is a high efficient TA cloning kit. The kit can be applied to theTA cloning of blunt-end PCR products amplified using KOD -Plus- [Code No.
KOD-201] or KOD FX [Code No. KFX-101]. The kit contains pTA2 Vector, 2x
Ligation Buffer, T4 DNA Ligase and 10x A-attachment Mix.
10 x A-attachment mix is a reagent comprising anti-KOD DNA polymerase antibody
specific to KOD 3’J5’ exonuclease activity (proof-reading activity), as well as Taq
DNA polymerase, which exhibits terminal transferase activity. PCR products from
KOD -Plus- [Code No. KOD-201] and KOD FX [Code No. KFX-101] possess bluntends due to 3’J 5’ exonuclease activity of the KOD DNA polymerase. The 10 x
A-attachment mix allows for PCR products to acquire overhanging dA at the 3’-ends.
Products with 3’-dA overhangs can be directly cloned into arbitrary T-vectors using
ligation reagents, such as Ligation high Ver.2 [Code No. LGK-201].

KOD DNA polymerase

Anti-KOD DNA polymerase
antibodyInhibition of theproof-readingacitivity

PCR productAA

Addition of dA

Taq DNA polymerase

Fig.

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Principle of the 10 x A-attachment mix

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NOTICE TO PURCHASER:

LIMITED LICENSE

Use of this product 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 productincludes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for thepurchaser’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 fromRoche. 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.

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TOYOBO Bio-Technology, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

7NOT FOR HUMAN OR DIAGNOSTIC USE.