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F0939K

Instruction manual KOD Dash 2109

KOD Dash
LDP-101

250 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]

Related products[10] References

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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Tel(81)-6-6348-3888
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TOYOBO Bio-Technology, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY.

1NOT FOR HUMAN OR DIAGNOSTIC USE.

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

Introduction

Description
KOD Dash is a highly efficient DNA polymerase mixture developed based on the Barns’
method1). This method uses a DNA polymerase which lacked a 3’5’ exonuclease(proofreading) activity and a small amount of an archaeal DNA polymerase with
proofreading activity. In the reagent, the 3’5’ exonuclease activity-deficient mutant<KOD EXO(-)> of KOD DNA polymerase2) and KOD DNA polymerase are used.
Because the proofreading activity repairs misincorporated nucleotide bases causing thetermination of a polymerase reaction, PCR with this mixed enzyme solution enables
highly efficient amplification.
KOD Dash generates dA overhang-ended PCR products. Therefore, the PCR products
can be cloned using a standard TA cloning method.

Features-This enzyme mixture is effective for the amplification of various targets from a small
template amount. The elongation ability of this enzyme mixture is much greater than thatof the normal Taq DNA polymerase.
-This enzyme mixture shows greater elongation velocity than Taq DNA polymerase (2folds) and Pfu DNA polymerase (6 folds) due to the property of KOD DNA polymerase.
-The PCR error ratio of this enzyme mixture is approximately 3 to 4 times less than thatof Taq DNA polymerase.

repairing
3’
5’

3’
5’

DNA polymerase(lacking proofreading activity)

DNA polymerase(having proofreading activity)

missincorporation
3’5’pausingDNA polymerase(lacking proofreading activity)

3’5’DNA polymerase(lacking proofreading activity)

Fig. 1. The principles of the Barns’ technology.

[2]Components

The provided reagents include the following components for 200 reactions:

KOD Dash (2.5 U/l)
10x PCR Buffer for KOD Dash*
2 mM dNTPs

100 l1.2 ml
1.0 ml*The final Mg concentration in the reaction mixture is 1.2 mM.

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

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

FOR RESEARCH USE ONLY.

1NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 3 ] Quality Testing[ 4 ] Primer Design[ 5 ] Cloning ofPCR products[ 6 ] Protocol

A quality check was performed by amplifying the human -globin gene (8.5 kb) and thelambda phase gene (12 kb).

PCR primers should be designed according to general guidelines. For the amplification ofa long target (≥ 4 kb), the melting temperature (Tm) of the primers should be set over72 °C.

The PCR products obtained with amplification by KOD Dash can be cloned according toa standard TA-cloning method.

1. Standard reaction setup
The following procedures have been designed for use with the components provided inthis kit. Before preparing the reaction mixture, all components should be completely
thawed, except for the enzyme solution.
*10x Buffer may precipitate. If precipitation occurs, dissolve it completely byoverturning and mixing before use. The precipitate on the bottom of the tube can beeasily dissolved by tapping it and then mixing it with the solution. If the precipitate isdifficult to dissolve, repeat both heating the reagent at 50℃ for 5 min and mixing it.
Before preparing the reaction solution, mix each reagent thoroughly before use.

Component
10x Buffer for KOD Dash
2 mM dNTPs*
10 pmol/l Primer #1
10 pmol/l Primer #2

Volume
5 l
5 l
1 l
1 l
X l

Template DNA
PCR grade water
KOD Dash (2.5 U/l)
Total reaction volume

Y l
0.5 l50 lFinal Concentration
1x0.2 mM each
0.2 M
0.2 M
Genomic DNA 10-1000 ng/50 l
Plasmid DNA 1-50 ng/50 lcDNA ≤1 g （RNA equiv.)/50 lE. coli cells (small amount)
1.25 U / 50 l* Do not use dNTPs from other kits or companies.
Notes:-Thin-wall tubes are recommended for PCR use. A total reaction volume of 50 l isrecommended.
-For amplification of a long target (≥ 8 kb), the use of a final concentration of 0.35 mM
dNTPs may reduce non-specific amplification.
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Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
bio_overseas@toyobo.jp

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

FOR RESEARCH USE ONLY.

2NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio

2. Cycling conditions
The following cycling steps are recommended.
(1) Cycling conditions for < 4kb targets.
< 3-step cycle >Pre-denaturation

94 °C, 0-2 min

Denaturation

94 °C, 30 sec.

Annealing

Tm-[0-5] oC*, 1-5 sec.

Extension

74 °C, 30 sec./kb

25-35 cycles*Tm value of the primer minus 0°C-5°C(2) Cycling conditions for ≥ 4kb products.
< 2-step cycle >Pre-denaturation

94 °C, 0-2 min

Denaturation

94 °C, 30 sec.

Extension

68-70 °C, 30 sec./kb

25-35 cycles

Note : If the Tm value of the primer is under 70 °C, the 3-step cycle is recommended.
Notes:-Extension time should be set at 30 sec. per 1 kb of target length.
-For colony-direct PCR, the pre-denaturation step should be set for 4 min.

[7]

ExamplesExample 1．Colony-direct PCR using E. coli cells.

M1

2

3

44.5 kb

Template:Small amount of E. coli cells (JM109)
from colonies
Insert: Transferrin receptor 4.5 kb
Vector: pBluescript II
PCR cycling condition:

Lane 1: Insert (+)
Lane 2: Insert (-)
Lane 3: Insert (+)
Lane 4: Insert (+)

94 ℃ 4 min.
98 ℃ 30 sec.
60 ℃ 5 sec.
72 ℃ 2.5 min.

30 cycles

F primer: TCGAGGTCGACGGTATCGAT (20mer GC=55%)
R primer: CGCTCTAGAACTAGTGGATC (20mer GC=50%)*Primers are designed on the vector.

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

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

FOR RESEARCH USE ONLY.

3
NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 8 ] Troubleshooting
Symptoms

Cause
Cycling conditions are not
optimal

No PCR product / low yield

Smearing / extra band(s)Primers are not good
Too much E. coli cells(Colony direct PCR)
Cycling conditions are not
optimalSolutions
Lower annealing temperature increments to a
maximum of Tm-10 °C.
Increase the number of cycles by 2-5 cycles.
Check the design and/or quality of primers.
Decrease the amount of E. coli cells from colonies.
Decrease the number of cycles by 2-5 cycles.

[ 9 ] Related products
Product nameHighly efficient cDNA synthesis kit

Package
100 rxns

Code No.
FSK-101

10,000 U

TRT-1012,500 U
250 U

SIN-201
BTQ-101

250 U

BTQ-201

200 U

KOD-201

200 U

KFX-101

ReverTra Ace® -Highly efficient reverse transcriptase

ReverTra Ace®
RNase inhibitor (Recombinant type)
Highly efficient Taq DNA polymerase

Blend Taq®
Highly efficient Taq DNA polymerase (Hot start version)

Blend Taq® -PlusHigh fidelity PCR enzyme

KOD -PlusHighly reliable PCR enzyme

KOD FX[ 10 ]

References1) Barns WM, Proc. Natl. Acad. Sci. USA, 91: 2216-2220 (1994)
2) Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M,and Imanaka T, Appl Environ Microbiol., 63: 4504-10 (1997)

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

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

FOR RESEARCH USE ONLY.

4NOT FOR HUMAN OR DIAGNOSTIC USE.