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F0939K

Instruction manual KOD Dash 0810

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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TOYOBO Bio-Technology, CO., LTD.
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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’J5’ exonuclease(proofreading) activity and a small amount of an archaeal DNA polymerase with
proofreading activity. In the reagent, the 3’J5’ 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/μl)
10x PCR Buffer for KOD Dash*
2 mM dNTPs

100 μl1.2 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
tech_osaka@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 β-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.

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

Volume
5 μl
5 μl
1 μl
1 μl
X μl

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

Y μl
0.5 μl50 μl

Final Concentration
1x0.2 mM each
0.2 μM
0.2 μM
Genomic DNA 10-1000 ng/50 μl
Plasmid DNA 1-50 ng/50 μl
cDNA ≤1 μg （RNA equiv.)/50 μl
E. coli cells (small amount)
1.25 U / 50 μl* Do not use dNTPs from other kits or companies.
Notes:-Thin-wall tubes are recommended for PCR use. A total reaction volume of 50 μl is
recommended.
-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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TOYOBO Bio-Technology, CO., LTD.
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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 ] Examples

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

M1234

4.5 kbTemplate: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
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tech_osaka@toyobo.jp

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

FOR RESEARCH USE ONLY.

3NOT 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 namePackage
100 rxns

Code No.
FSK-101

10,000 U

TRT-1012,500 U
250 U

SIN-201
BTQ-101

Highly efficient Taq DNA polymerase (Hot start version)
®

250 U

BTQ-201

High fidelity PCR enzyme

200 U

KOD-201

200 U

KFX-101

Highly efficient cDNA synthesis kit®

ReverTra Ace -α-Highly efficient reverse transcriptase®

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

Blend Taq

Blend Taq -PlusKOD -Plus-

Highly reliable PCR enzyme

KOD FX[ 10 ] References
1) 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
tech_osaka@toyobo.jp

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

FOR RESEARCH USE ONLY.

4NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio

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.

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

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

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.