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F1066K

Instruction manual KOD -Plus- Neo 2004

KOD -Plus- Neo
KOD-401

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. Do not use for diagnostic or clinical purposes. Please observe
general laboratory practices and safety precautions while using this kit.

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

Description
KOD -Plus- Neo is based on a DNA polymerase from the hyperthermophilic Archaeon
Thermococcus kodakarensis KOD11) 2). This polymerase exhibits excellent PCR fidelity
because of its efficient 3’5’ exonuclease activity (proof reading activity). This productcontains a unique “elongation enhancer” that suppresses the “plateau effect” produced byconventional PCR. Therefore, this reagent exhibits greater amplification efficiency andelongation capability compared to the previous version of KOD -Plus- (Code No. KOD201).
Moreover, this enzyme requires only 30 sec/kb for the PCR extension step. This facilitates
the long range PCR.
This enzyme contains two anti-KOD DNA polymerase antibodies that inhibit polymerase
and 3’5’ exonuclease activity, thus allowing for Hot Start PCR3). This polymerase
generates blunt-end PCR products due to 3’5’ exonuclease (proof-reading) activity.

Features-KOD -Plus- Neo exhibits 80-fold greater PCR fidelity than Taq DNA polymerase.
-“Elongation enhancer” enables greater amplification efficiency and elongation capability(up to 24 kb from human genomic DNA) compared to conventional PCR.

-Requires only 30 sec/kb for the PCR extension step.
-2-step cycle conditions can be used for amplification using ≥ 20 mer primers (melting
temperatures, Tm >63°C).

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www.toyobo.co.jp/e/bio*The Nearest Neighbor method is recommended to calculate the Tm of primers. The Tm values inthis manual were calculated using this method with the following parameters.
Na+ concentration: 50 mM
Oligonucleotide concentration: 0.5 µM[ 2 ] Components

KOD -Plus- Neo (1.0 U/μL)*

200 μL x 1

10 x PCR Buffer for KOD -Plus- Neo1 mL x 1

25 mM MgSO4

1 mL x 1

2 mM dNTPs

1 mL x 1*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymeraseand 3’5’ exonuclease activity.

[3]

Quality Testing

Quality control was performed by amplifying the human β-globin gene (17.5 kb).

[4]

Primer Design-Primers should be 22–35 bases with Tm >63°C.
-Optimal GC content of primers is 45–60%. Ideal GC contents of the 5’ half and the 3’ halfare 60–70% and 40–50%, respectively.
-Primers for long target amplification should be 25-35 bases with Tm >65°C.
-Primers containing inosine cannot be used.
-The Tm of primers should be calculated using the Nearest Neighbor method. The Tmvalues in this manual were calculated using this method with the following parameters.
Na+ concentration: 50 mM
Oligonucleotide concentration: 0.5 µM[ 5 ] Cloning ofPCR products-KOD -Plus- Neo generates blunt-end PCR products due to its 3’5’ exonuclease (proofreading) activity. Therefore, PCR products can be cloned according to blunt-end cloningmethods.
-PCR products of KOD -Plus- Neo should be purified prior to restriction enzyme treatments.
The 3’5’ exonuclease activity of KOD DNA polymerase remains at the end of the PCR
reaction.
-The dedicated TA cloning kit “TArget clone™ -Plus- (Code No. TAK-201)” isrecommended for the cloning of blunt end PCR products produced by KOD DNA
polymerase (see [10] Related product).

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www.toyobo.co.jp/e/bio[ 6 ] Protocol

1. Standard reaction setup
The following procedure is designed for use with the components provided in this kit.
Before mixture preparation, all components should be completely thawed, except for theenzyme solution.
Component
Volume10x Buffer for KOD -Plus- Neo
5 μL
2 mM dNTPs*
5 μL
25 mM MgSO43 μL
10 pmol/μL Primer #1
0.75–1.5 μL
10 pmol/μL Primer #2
0.75–1.5 μL
Template DNAX μL

PCR grade water
KOD -Plus- Neo (1.0 U/μL )Total reaction volume

Y μL
1 μL
50 μL

Final Concentration
1x0.2 mM each
1.5 mM
0.15–0.3 µM
0.15–0.3 µM
Genomic DNA ≤ 200 ng/50 μL
Plasmid DNA ≤ 50 ng/50 μL
cDNA ≤ 200 ng（RNA equiv.)/50 μL
1.0 U/50 μL*Do not use dNTPs from other kits or companies.
Notes:-Optimal primer concentration is 0.3 µM. In the case of long target (≥10 kb), a reduced
primer concentration (0.15 µM) may give more effective amplification.
-The addition of DMSO (final conc. 2–5%) is beneficial for the amplification of GCrich targets. Decreased PCR fidelity does not occur in the presence of DMSO (See step2, Cycling conditions).
-Contaminating RNA in cDNA or genomic DNA inhibits the PCR reaction by chelating
Mg2+. PCR should be performed using template DNA containing <200 ng RNA.
-Quality of template DNA should be checked by electrophoresis. The length and purityof template DNA affects amplification results.
-Templates containing uracil cannot be used for amplification.
-For PCR reactions, thin-wall tubes are recommended. A total reaction volume of 50 μL
is also recommended.

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2. Cycling conditions [Important]Two-step cycle conditions with primers ≥20 mer, Tm >63°C are recommended for
effective amplification using KOD -Plus- Neo.

A. 2-step cycle
If the Tm value of the primer is over 63°C, a 2-step cycle is recommended.
< 2-step cycle >Pre-denaturation:

94°C, 2 min.

Denaturation:

98°C, 10 sec.

Extension:68°C, 30 sec./kb

25-45 cycles

Notes:-For amplification using low copy templates or amplification of long targets (>10 kb),longer extension times (up to 1min/kb) or higher Mg concentrations (up to 2 mM
final concentration) may increase the yield.
-The addition of DMSO (final conc. 2–5%) may be beneficial for the amplification ofGC-rich targets. The concentration of DMSO used should be determined according
to the Tm of the primers.
<25 mer≥25 meroror

Tm <68°C: up to 2%
Tm ≥68°C: up to 5%-In the case of amplification failure a 3-step cycle may be required.

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B. 3-step cycle
When the Tm of primers are less than 63°C, a 3-step cycle should be used.
< 3-step cycle >Pre-denaturation:

94°C, 2 min.

Denaturation:

98°C, 10 sec.

Annealing:[Tm]oC, 30 sec.

Extension:

68°C, 30sec./kb

25-45 cycles

Notes:-Amplification using low copy templates or amplification of long targets (>10 kb) may
be improved by increasing the extension time (up to 1 min/kb) or Mg concentration(up to 2 mM final concentration).
-The addition of DMSO (final conc. 2–5%) might be beneficial for the amplification ofGC-rich targets.

C. Step-down cycle
If non-specific amplification is observed with 2-step and 3-step cycle conditions (extraor smeared bands observed after electrophoresis of PCR product), then the step-downcycle may improve specificity.

< Step-down cycle >Pre-denaturation:

94°C, 2 min.

Denaturation:

98°C, 10 sec.

Extension:

74°C , 30 sec./kb

Denaturation:

98°C, 10 sec.

Extension:

72°C , 30 sec./kb

Denaturation:

98°C, 10 sec.

Extension:

70°C , 30 sec./kb

Denaturation:

98°C, 10 sec.

Extension:68°C , 30 sec./kb

Extension:

68°C , 7 min.

5 cycles5 cycles5 cycles15-30 cycles

Notes:-Amplification from low copy templates or amplification of long targets (>10 kb), may
be improved by increasing the extension time (up to 1 min/kb) or the Mg concentration(up to 2 mM final concentration).

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www.toyobo.co.jp/e/bio-The addition of DMSO (final conc. 2–5%) might be effective for the amplification ofGC-rich targets. The DMSO concentration should be determined according to theTm of primers.
<25 mer≥25 mer[ 7 ] Examplesoror

Tm <68°C: up to 2%
Tm ≥68°C: up to 5%

Performance data 1． PCR fidelity
Mutation frequency was measured by sequence analysis of human β-globin gene products
amplified from human genomic DNA via TA cloning with TArget Clone™ -Plus-. KOD Plus- Neo showed excellent fidelity and the mutation frequency was equal to that of theprevious version of the enzyme (KOD -Plus-).

Performance data 2． Elongation capability
Targets of various sizes were amplified from human genomic DNA by several PCR
enzymes according to the recommended conditions of each enzyme. KOD -Plus- Neo
successfully amplified targets up to 17.5 kb.

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Performance data 3． Amplification from low copy templates
Four genes were amplified using 0.5 ng cDNA template (RNA equiv.). Templates weresynthesized from total RNA of HeLa cells. KOD -Plus- Neo successfully amplified allgenes.

Performance data 4． Elongation rate
The β-globin gene (3.6 kb) was amplified from human genomic DNA (50 ng) using various
extension times. KOD -Plus- Neo can amplify a 3.6 kb target using an extension time of30 sec/kb.

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Application data 1． Amplification of various protein kinase targets
Various protein kinase open reading frames (ORFs) were amplified using cDNA
synthesized from total RNA of HeLa cells. KOD -Plus- Neo successfully amplified alltargets.

Application data 2． Amplification of long targets
Long targets were amplified from human genomic DNA using various concentrations ofprimers. Excessive amounts of primer inhibit the amplification. Therefore, long targets(>10 kb) should be amplified using lower primer concentrations of approximately 0.15 µM.

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

Cause

Solution

Cycling conditions are notsuitable.

Using the 3-step cycle, lower annealing
temperature incrementally to a maximum of Tm-5–10°C.
Prolong the extension time to 1 min./kb.
Increase the number of cycles by 2–5 cycles.
Increase the Mg concentration up to 2 mM.
Add DMSO 2–5%.
[See Cycling conditions]
Decrease the primer concentration incrementally
down to 0.15 µM.
Use fresh primers.
Redesign primers.
Check the quality of template DNA. RNA inhibits
amplification.
Increase the amount of template DNA.
Increase enzyme concentration up to 1.5–2.0 U/50
μL .
Decrease the number of cycles by 2–5 cycles.
Change from 3-step cycle to 2-step cycle.
Change from 2-step cycle to Step-down cycle.
Use fresh primers.
Redesign primers.
Reduce the amount of template DNA.
Reduce MgSO4 incrementally down to 1.0 mM.
Reduce enzyme concentration to 0.5–0.8 U/50 μL .
Clone the PCR products according to general bluntend cloning guidelines.
Use TArget clone™ -Plus- (Code No.TAK-201)[See Related products].

Mg concentration is low.
High GC content of target
sequence.
Quality and/or quantity ofprimers is not sufficient.

No PCR product/low yield

Quality and/or quantity oftemplate DNA is notsufficient.
Enzyme concentration islow.
Cycling conditions are notsuitable.
Smearing/extra band

Poor TA cloning efficiency.

[ 9 ] Reference1)

Quality of primers is notsufficient.
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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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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