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F0934K

Instruction manual KOD -Plus- 2004

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

CAUTIONAll reagents in this kit are intended for research purposes only. Not for diagnostic or clinical use. Please observe generallaboratory safety precautions while using this kit.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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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 kodakarensis 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’5’ exonuclease activity, thus
allowing for Hot Start PCR3). KOD -Plus- generates blunt-end PCR products, due to 3’5’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’5’ exonuclease (proof-reading) activity. The PCR
error rate of KOD -Plus- is approx. 80 times less than Taq DNA polymerase.

Table. 1

PCR fidelity comparison of each PCR enzyme.

Colony number
MutantTotal
10,6101068
10,9006,520
7610,560
780

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

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 baseassay with the rpsL gene 4).

[ 2 ] Components

This kiy includes the following components for 200 reactions:
KOD -Plus- (1.0 U/μL) *10× Buffer for KOD -Plus25 mM MgSO42 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’5’ exonuclease activity.

[3]

Quality TestingJAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
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tech_osaka@toyobo.jp

Quality checks are performed by amplifying the β-globin gene (3.6 Kb) and p53 gene (4.0Kb).

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

1

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’5’ exonuclease (proof- reading)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’5’ 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 μL

Template 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 dimethyl sulfoxide (DMSO; final conc. 2-5%) might be effective foramplification of GC-rich targets. No decrease in PCR fidelity has been observed using
DMSO.
-Contaminating RNA inhibits the PCR reaction by chelating Mg2+. PCR should be
performed using template DNA containing <100 ng of RNA.

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2www.toyobo.co.jp/e/bio

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.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
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CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

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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.
The P53 gene (4 kb) was amplified by KOD-Plus-Ver.2 and high fidelity PCR enzymes
from other companies using human genomic DNA as the template. KOD -Plussuccessfully amplified the target genes without generating primer-dimmers.

M123456M

Template: Human genomic DNA
1,3,5: 50ng, 2,4,6: 100ng
Target: p53 gene 4kb
M: l/Hind III 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.
The TGF-b gene (2kb, GC%=70) was amplified using KOD-Plus-Ver.2 with or withoutDMSO. The addition of 2-3 % DMSO permitted effective amplification of the target.

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.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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www.toyobo.co.jp/e/bio[ 8 ] TroubleshootingSymptom

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.

No PCR product/low yield

Quality and/or quantity oftemplate DNA is notsufficient.
Cycling condition is notsuitable.
Primer is not good.
Smearing/extra band
Too much template DNA
Too much Mg
Too much enzyme
PCR products have bluntends.

Poor TA cloning efficiency1)[ 9 ] References

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, J
Mol 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.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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www.toyobo.co.jp/e/bio[ 10 ] Related products
Product name
TArget Clone™ -Plus10x A-attachment mix
Ligation high Ver.2

Package
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 the TA cloning ofblunt-end PCR products amplified using KOD -Plus- [Code No. KOD-201], KOD -Plus- Neo [CodeNo. KOD-401] 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’5’ 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 blunt ends due to 3’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].

Fig.

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Principle of the 10 x A-attachment mix

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