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Instruction manual KOD EXO(-) 2004

KOD exo(-) DNA Polymerase

250 U 200 reactions

Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Protocol
1. Standard reaction setup
2. Cycling conditionJAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
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tech_osaka@toyobo.jp[4]

Example[5]

References

CHINA
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Tel (+86)-21-58794900
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FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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

Description

KOD exo(-) is a 3’5’ exonuclease minus mutant developed based on KOD DNA
polymerase from a hyperthermophilic Archaeon Thermococcus kodakaraensis KOD11) 2).
KOD exo(-) shows excellent elongation ability and PCR efficiency.

Features-A fast mode cycle can be applied.

[ 2 ] Components

This reagent includes the following components;
KOD exo(-) (2.5 U/μL)
10× Buffer*
2mM dNTPs*The buffer contains 12mM MgCl2.

[3]Protocol
1. Standard reaction setup
The following procedure is designed for use with the components provided in this kit.
Before preparing the mixture, thaw all components completely except for the enzyme
solution.
Component
10x buffer for KOD exo(-)*
2mM dNTPs**
10pmol /μL Primer #1
10pmol /μL Primer #2Template DNA

Volume5 μL
5 μL l0.5~1μL
0.5~1μL
X μL

Final Concentration
1×
0.2 mM each
0.1~0.2 µM
0.1~0.2 µM
Genomic DNA ~1000 ng / 50 μL
Plasmid DNA ~50 ng / 50 μL
cDNA ~200 ng （RNA equiv.) / 50 μL

PCR grade water
Y μL
KOD exo(-) (2.5 U/μL )0.5 μL1.25 U / 50 μL
Total reaction volume50 μL*The buffer contains 12mM MgCl2. For the fast mode cycle, an extra MgCl2 should beadded up to 7.2mM (final concentration).

** Do not use dNTPs from other kits or companies

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

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900
www.bio-toyobo.co.jp

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/bio

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 of GC-richtargets. Decreased PCR fidelity has been confirmed to not take place with DMSO.
-Contaminated RNA (used for cDNA) or genomic DNA inhibits the PCR reaction bychelating Mg2+. PCR should be performed using template DNA containing <200 ng RNA
component.
-Primers should be 22~34 bases, with a melting temperature (Tm) > 60°C. PCR primers
should be designed according to the general guidelines.

2. Cycling condition
The following cycling steps are recommended.
(1)Normal cycle (3-step cycle)< 3-step cycle >Pre-denaturation :94°C, 2min.

Denaturation :94°C,15sec.

Annealing :(Tm~Tm-5) °C, 30 sec.

Extension :72~74 °C, 30 sec. /kb

25~35 cycles(2)Normal cycle (2-step cycle)
This cycle is suitable for the amplification >4kb targets.
< 2-step cycle >Pre-denaturation :94 °C, 2min.

Denaturation :94 °C,15sec.

Annealing :68~70 °C, 30 sec. /kb

25~35 cycles(3)Fast mode cycleThis cycle can be applied to <1.5kb targets. In this cycle, an extra MgCl2 should be added
up to 7.2 mM (final concentration).
< 3-step cycle >Denaturation :95 °C, 0 sec.

Annealing :(Tm~Tm-5) °C, 0 sec.

Extension :72°C, 10~15 sec./kb

25~35 cycles[ 4 ] Example

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

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900
www.bio-toyobo.co.jp

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/bio

Example 1. Fast mode cycle (1)Example 2. Fast mode cycle (2)[ 5 ] References

1)

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)

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

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900
www.bio-toyobo.co.jp

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.