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F1440K

Instruction manual for KOD -Multi & Epi- 2004

KOD -Multi & Epi-TM
KME-101

200 U

200 reactions

Store at −20°C

Contents[1]

Introduction[2]

Components[3]

Primer design[4]Template[5]

Cloning of PCR products[6]

Protocol
1. Singleplex PCR
2. Multiplex PCR
3. PCR using bisulfite-treated DNA[7]

Troubleshooting[8]

References[9]

Related products

CAUTIONAll reagents in this kit are intended for research purposes only. Do not use for diagnostics or clinical purposes. Please observe generallaboratory precautions and safety measures while using this kit.
.
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1

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

Description
KOD -Multi & Epi-TM is a high-fidelity PCR enzyme based on genetically modified KOD
DNA polymerase1,2) (UKOD). This modified enzyme enables amplification from templates
containing uracils (U) or using primers containing inosines (I) and uracils (U). Furthermore,
addition of the Elongation Accelerator significantly reduces amplification bias during PCR.
KOD -Multi & Epi-TM can be applied to various purposes such as a) multiplex PCR, b)bisulfite PCR in epigenetics research, and c) metagenomics research. This enzyme is also
applicable for the preparation of DNA fragments for next-generation sequencing andcapillary sequencing via cloning because the enzyme exhibits about 11-fold higher PCR
fidelity.
KOD -Multi & Epi-TM contains two types of anti-KOD DNA polymerase antibodies that
inhibit the polymerase and its 3ʹ5ʹ exonuclease activity, thus allowing for Hot Start PCR3).
Furthermore, KOD -Multi & Epi-™ generates blunt-end PCR products because of its
3ʹ5ʹ exonuclease (proof-reading) activity.

Features
Homogeneous amplification (Low bias)
Homogeneous multiplex PCR of targets up to 10 kb can be achieved. Various regions of
the genome or transcriptome can be amplified homogeneously even if these regions
contain GC bias. The resulting amplicons are also suitable for next-generation sequencing
analyses.
-

Effective amplification from templates containing uracils (U)Effective amplification can be achieved using bisulfite-treated template DNA that
contains uracils (U).

-Primers containing uracil (U) or Inosine (I) can be used
KOD -Multi & Epi-TM can use primers containing inosines (I) or uracil (U), whereas
conventional high-fidelity PCR enzymes cannot.

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High fidelity
KOD -Multi & Epi-TM exhibits approximately 11-fold higher fidelity than Taq DNA
polymerase. The enzyme can be used for various purposes where this would be anadvantage (e.g. in the preparation of long target amplicons for sequencing).

-

Applicable for amplification from crude samples
KOD -Multi & Epi-TM can be used for amplification from various crude samples (e.g.
blood, lysates from animal or plant sources, soil, food, etc.) because of its tolerance of the
PCR inhibitors commonly present in crude samples.

-

Highly efficiency
PCR efficiency is improved by adding an “Elongation Accelerator”. The extension rate
can be reduced by decrements of up to 15 s/kb in singleplex PCR. (Note that to realize
sufficient amplification, the extension rate of ~30–60 s/kb is recommended for
amplifications using bisulfite-treated DNA or for the multiplexed amplification of longtargets.)

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www.toyobo.co.jp/e/bio[2] Components

KOD -Multi & Epi-TM (1.0 U/µL)*
2× PCR Buffer for KOD -Multi & Epi-TM**

200 µL × 1
1.7 mL × 3*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize
the polymerase and inhibit its 3ʹ5ʹ exonuclease activities.
**The 2× PCR Buffer for KOD -Multi & Epi-TM contains dNTPs (dATP, dGTP, dCTP,
dTTP), and Mg2+ at 4.0 mM (final concentration: 2.0 mM).

[3] Primer Design(1) Singleplex PCR-

Primers should be 22–35 bases with a melting temperature (Tm) over 60°C.

-

Optimal GC content of the primers is 45–60%. The ideal GC content of the 5ʹand 3ʹ-ends of the primers is 60–70% and 45–50%, respectively.

-

The priming efficiency of the primers can be promoted by anchoring the 3ʹ-endof the primers with a G or C.

-

Primers should be designed so as not to generate intramolecular secondary
structures or primer dimers.

-

Primers for long target amplification should be 25–35 bases with Tm over 65°C.

-

Primers containing inosine (I) or uracil (U) can be used and are applicable formany purposes such as metagenomic analyses.

-

The Tm of primers should be calculated using the nearest neighbor method. The
Tm values in this manual were calculated using this method with the following
parameters: Na+ concentration, 50 mM and oligonucleotide concentrations, 0.5μM.

(2) Multiplex PCR

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Primers should be designed according to the concepts described in (1).

-The performance of each primer set should first be confirmed by singleplex PCR
using the same PCR cycle plan to be used later for multiplex PCR.

-

The difference in Tm between primer pairs should be as small as possible.

-

Different primer sets should not exhibit cross-complementarity between targets
in the same multiplex PCR reaction.

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www.toyobo.co.jp/e/bio(3) PCR for bisulfite-treated DNA-

Primers should be designed according to the same basic concepts described in(1). The design of primers can be facilitated by the use of specific software thatallows the replacement of cytosine residues with uracil.

-

The following online tool is recommended:
MethPrimer, http://www.urogene.org/methprimer/index.html[4] Template
The following table indicates the applicable samples and appropriate amounts for PCR (50µL reaction):
Sample type

Appropriate amountTypical amount

Genomic DNA: Eukaryotic
Genomic DNA: Prokaryotic
Plasmid DNA
cDNA
Bisulfite-treated DNAb
Crude sample (blood, lysates, etc.)c

5–200 ng
0.1–100 ng
10 pg–50 ng~200 ng (RNA equiv.) a~200 ng

50 ng
10 ng
1 ng
50 ng (RNA equiv.)a

Excessive amounts of RNA tend to inhibit PCR. The amount of cDNA to be prepared per reaction
should be less than 200 ng (RNA equiv.).
b
Different commercially available bisulfite kits yield different conversion efficiencies and levels of DNA
degradation. Artificially methylated DNA can be used as a positive control to check the conversion
efficiency of the bisulfite treatment.
cVarious crude samples can be used as templates. For a detailed protocol for the use of crude samples,
please refer to the instruction manual for KOD FX Neo.
(http://www.toyobo-global.com/seihin/xr/lifescience/support/manual/KFX-201.pdf)[5] Cloning of PCR products-

KOD -Multi & Epi-TM generates blunt-end PCR products because of its 3ʹ5ʹ
exonuclease (proof-reading) activity. Therefore, the resulting PCR products can becloned using blunt-end cloning methods.

-

PCR products of KOD -Multi & Epi-TM should be purified prior to restriction enzyme
treatments in downstream cloning steps. The 3ʹ5ʹ exonuclease activity of KOD
DNA polymerase is present until the end of the PCR reaction.

The dedicated TA cloning kit, TArget clone™-Plus- (Code No. TAK-201), is recommended
for the cloning of blunt-end PCR products produced by KOD DNA polymerase (see [9]Related products).

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

Protocol

1. Singleplex PCR(1) Reaction set-up
Target genes up to 40 kb in length can be amplified from human genomic DNA using
singleplex PCR.
Component

Volume

PCR-grade water

X μL

Final Concentration

2× PCR buffer

25 μL

1×

10 pmol/μL Primer #1

1.5 μL0.3 μM

10 pmol/μL Primer #2

1.5 μL0.3 μM

Template DNA

Y μL

Genomic DNA ≤ 200 ng/50 μL

for KOD -Multi Epi-TM*

Plasmid DNA ≤ 50 ng/50 μL
cDNA ≤ 200 ng (RNA equiv.)/50μL
Crude sample ≤ 5 μL/50 μL
KOD -Multi & Epi-TM

1 μL

Total reaction volume

50 μL

1.0 U/50 μL*2× PCR buffer for KOD -Multi & Epi-TM contains dNTPs (dATP, dGTP dCTP, dTTP),
and 2 mM Mg2+.

Notes:-

When non-specific or smeared bands are observed, the primer concentration
should be decreased in decrements of up to 0.1 µM (final).

-

In cases where degenerate primers are used, the primer concentrations can beincreased up to 3.0 µM (final) depending on the degree of degeneracy.

(2) Cycling conditions
The 3-step cycle can be applied in cases where the Tm of the primers are ≤ 65°C. A 2step cycle can be applied in cases where the Tm of the primers are > 65°C.

3-step cycle [In cases where primers’ Tm ≤ 65°C]
Pre-denaturation:

94°C, 2 min

Denaturation:

98°C, 10 s

Annealing:

Tma, 10 s

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68°C, X s

30 cyclescb

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2-step cycle [In cases where primers’ Tm > 65°C]
Pre-denaturation:

94°C, 2 min.

Denaturation:

98°C, 10 s

Extension:

68°C, X sb

30 cyclesc

Notes:
m−5°C to enhance
amplification yield.
bThe extension time should be determined according to the following table.
aThe annealing temperature can be decreased in decrements of up to T

Extension time

Purified DNA (Genomic DNA, PlasmidDNA, cDNA)
Target does not
Targetcontain GC
contains GC
cluster*cluster*
15–30 s/kb#
30 s/kb

Crude sample(Tissue, lysate),

60 s/kb*GC cluster: a GC-rich region with GC content ≥ 70%.
#
The extension time should be ≥ 30 s/kb in the case of a 2-step cycle or when using
degenerate primers. In cases where low copy numbers of the template DNA are present
or long targets over 10 kb require amplification, the yield can be increased by setting theextension time to 60 s/kb.
cThe PCR cycle number can be increased up to 40 cycles.

2.

Multiplex PCR(1) Reaction set-up
Multiple targets of up to 10 kb can be stably amplified from human genomic DNA.

Component

Volume

PCR-grade water

X µL

Final Concentration

2× PCR buffer

25 µL

1×

Y µL

0.3 µM each#for KOD -Multi & Epi-TM*
Primers

Genomic DNA ≤ 200 ng/50 µL
Template DNA

Z µL

Plasmid DNA ≤5 0 ng/50 µL
cDNA ≤ 200 ng （RNA equiv.)/50 µL
Crude sample ≤ 5µL50 µL

KOD -Multi & Epi-TM

1 µL

1.0 U/50 µL(1.0U/µL)*
Total reaction volume

50 µL*2× PCR buffer for KOD -Multi & Epi-TM contains dNTPs (dATP, dGTP dCTP, dTTP), and 2
mM Mg2+.

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

The primer concentrations should be optimized using the following guidelines:
Primer concentrations should be decreased in decrements of up to 0.1 µM if non-specificor smeared bands are observed.
Amplification bias in multiplex PCR can be improved by increasing the concentration ofprimers that yield relatively weak bands, or by decreasing the concentration of primers thatexhibit excessive amplification.
Commercially available primer sets for multiple targets should be used according to the
manufacturer’s instructions.

(2) Cycling conditions
The 3-step cycle can be applied in cases where the Tm of the primers are ≤ 65°C. The 2step cycle can be applied in cases where the Tm of the primers are > 65°C.
3-step cycle [In cases where primers’ Tm ≤ 65°C]
Pre-denaturation:

94°C, 2 min.

Denaturation:

98°C, 10 s

Annealing:

Tma, 30 s

Extension:

68°C, X sb

25 cyclesc

2-step cycle [In cases where primers’ Tm > 65°C]
Pre-denaturation :94°C, 2 min

Denaturation :98°C, 10 s

Extension :68°C, X sb

25 cyclesc

Notes:
aThe annealing temperature should be determined based on the primer with the lowest T .
m
The annealing temperature can be increased up to Tm+5°C to reduce non-specific orsmeared bands. To enhance amplification, it can be decreased in decrements up to Tm−5°C.
bThe extension time should be determined according to the following table:

Small targets (≤ 1 kb)

Large targets (1–10 kb)
Number of targets

≤ 10

> 10

≤ 10

30–60 s

30 s/kb*

30–60 s/kb*

30 s

30–60 s

30 s/kb*

30–60 s/kb*

60 s

60 s

60 s/kb*> 10<Purified DNA, cDNA> Extension times
3-step cycle
2-step cycle

15–30 s

<Crude samples> Extension times
3- and 2-step cycles*The extension time should be determined based on the largest target.
cThe PCR cycle number can be increased up to 40 cycles.

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60 s/kb*

www.toyobo.co.jp/e/bio(3) Application data
Targets from 1 to 10 kb were amplified by singleplex or multiplex PCR using the primer
pairs listed in the table below. Each target was successfully amplified by both singleplex
and multiplex PCR.

Primer
Chrome9_1 kb_F primer
Chrome9_1 kb_R primer
MSH6_1.8 kb_F primer
MSH6_1.8 kb_R primer
BRCA2_2.3 kb_F primer
BRCA2_2.3 kb_R primer
WT-1_2.5 kb_F primer
WT-1_2.5 kb_R primer
FANCE_3 kb_F primer
FANCE_3 kb_R primer
RAD51D_4 kb_F primer
RAD51D_4 kb_R primer
KRAS_5 kb_F primer
KRAS_5 kb_R primer
BRCA1_7 kb_F primer
BRCA1_7 kb_R primer
DDB2_10 kb_F primer
DDB2_10 kb_R primer

3.

Sequence
GAATTCCATATCTTTGCCAAACACTTGGTG
CCATGGGAAATGTGTTGAAGAAAACAAAGTGCAGAAGAGGAAGAAGAGATGGAGGTGGAGGTAAGAAGAGACAGGCAAAGT
CAGGTCTTAACCTAGCAGAGGAGGT
GGTTGGTCTGCCTGTAGTAATCAAGGAGGTCGAGCCACTCTTTATTACGTCTGACTCCCTTCGTCTAGTCTCTG
CAGTCTTCCGTTAGATATCCTGAGCCTTCTGCCTAGATCTCCAGAGGATTACAGTGAGACGTGAGACCCTATCTC
CACAAATCTATTGCCCTGATAGCATCTTCCTGTGGGCTAGAGATACACTG
CAAGCAACTAAGGTGAGTGGAAGAGGCCCTTTAAGCAAAGACAGTAGTCC
CATCTCTGTCTGGTCAATCCCTTACGCAGCAATAGTGGAAGACTGGTTACGCTAGAGCCACCATTAGACTCAGAC

Primer length
30
31
25
25
25
25
24
25
25
25
25
25
25
25
25
25
25
25

T m (℃)
72.1
73.5
65.9
65.7
65.7
65.5
65.7
66.0
64.9
65.8
65.7
65.7
65.9
66.0
65.8
65.7
65.7
65.2

GC(%)
40.0
38.7
48.0
48.0
52.0
48.0
50.0
52.0
48.0
48.0
52.0
40.0
52.0
48.0
48.0
48.0
48.0
52.0

Target size
1 kb
1.8 kb
2.3 kb
2.5 kb
3 kb
4 kb
5 kb
7 kb
10 kb

PCR using bisulfite-treated DNA(1) Reaction set-up
Targets up to 1.5 kb can be amplified using bisulfite-treated DNA.
Component

VolumeFinal Concentration

PCR grade water

X µL

2× PCR buffer

25 µL

1×

PrimersY µL

0.3 µM each#

Bisulfite-treated DNA

Z µL~200 ng/50 µL

KOD -Multi & Epi-TM (1.0U/µL)*

1 µL

1.0 U/50 µL

Total reaction volume

50 µL

for KOD -Multi & Epi-TM**PCR buffer for KOD -Multi & Epi-TM contains dNTPs (dATP, dGTP dCTP, dTTP), and 2 mM
Mg2+.
#

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The primer concentrations should be reduced step-wise in decrements of up to 0.1 mM (final).

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www.toyobo.co.jp/e/bio(2) Cycling conditions
The following 3-step cycle is recommended:

Pre-denaturation:

94°C, 2 min

Denaturation:

98°C, 10 s

Annealing:

Tma, 30 s

Extension:

68°C, X sb

40 cyclesc

Notes:aIn cases of poor amplification, the annealing temperature can be decreased to T −5°CmbThe extension time should be determined according to the following table:

Extension time

< 500 bp

500–1,000 bp≥ 1,000 bp

15 s

30 s

30 s/kb

In cases of poor amplification, the extension time can be extended up to two-fold.
cIf non-specific or smeared amplification bands are observed, the number of PCR cycles
should be reduced step-wise up to 30 cycles.

(3) Application data
The target DNAs (917–1,583 bp) were amplified using bisulfite-treated CpG-methylated
Jurkat genomic DNA. All targets were successfully amplified using KOD -Multi & Epi-TM.

【Bisulfite treatment】
EpiTect® Fast DNA Bisulfite Kit (QIAGEN)【Primer concentration】
0.3 µM eachM 1 2 3 4

【Sample】
55 ng (bisulfite-treated DNA)
/ 50 µl reaction【PCR cycle】
94℃, 2 min.
98℃, 10 sec.
60℃, 30 sec.
68℃, 30 sec./ kb
4℃, hold

Primer
TGFβ_917 bp_F
TGFβ_917 bp_R
BRCA2_1134 bp_F
BRCA2_1134 bp_R
APOE_1487 bp_F
APOE_1487 bp_R
TGFβ_1583 bp_F
TGFβ_1583 bp_R

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M

M. 100 bp DNA ladder
1. TGFβ 917 bp (AT content*1 : 69.9%)

1kb

2. BRCA2 1134 bp (AT content*1 : 63.9%)

500bp

3. APOE 1487bp (AT content*1 : 69.1%)
4. TGFβ 1583 bp (AT content*1 : 61.8%)

40 cycles

100bp*1 The AT content after bisulfite treatment are shown.

Sequence
TGGATTTTAAAGTTTTAGTTTTTTTTAGG
CAACTACTTCCAACCTCCCATAATAGGGGAATAGGTTTTGAGAGAATATTATACCACTAACCACATTAAACACTCGGTTTTTTTAAGTAGGGTGGTTTG
TCAAAAAACCAATTTCCTCTTTATCTTGGAGAAAGTTGATTTAGAGTTTG
CAACTACTTCCAACCTCCCATAATA

Primer length
29
25
25
25
24
25
25
25

T m (℃)
61.6
62.8
62.2
58.8
62.3
62.0
61.3
62.8

GC(%)
20.7
40.0
36.0
36.0
37.5
28.0
32.0
40.0

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Target size
917 bp1134 bp1487 bp1583 bp

www.toyobo.co.jp/e/bio[7] Troubleshooting(1) Singleplex PCR
Symptom

Cause

Solution

Cycling conditions are notsuitable.

Increase the extension time up to 60 s/kb.
Increase the number of cycles by 2–5 cycles.
Use a 3-step cycle instead of a 2-step cycle.
Lower annealing temperature in the 3-step cycling
procedure step-wise up to Tm−5°C.
In cases where degenerate primers are being used,
the annealing temperature should be decreased
step-wise up to Tm−5°C.
Increase the amount of template DNA.
Change the DNA purification method.
Use purified templates.
Decrease the amount of cDNA to reduce inhibition
by RNA contaminants.
Degrade or eliminate RNA in the cDNA sample.
Decrease the primer concentration step-wise from
0.3 µM to 0.1 µM.(This solution may be effective
for the amplification of targets over 10 kb.)
Primer concentration should be increased up to 3.0µM in cases where degenerate primers are being
used.
Use fresh primers.
Redesign primers.
Increase enzyme concentration to 1.5–2.0 U/50 µL.

Template DNA is of low
quality and/or quantity.

No PCR product/low yield

Poor primer performance.

Enzyme concentration is
too low.
Cycling conditions are notsuitable.
Too much template DNA.
Poor primer performance.
Smearing/Extra bands
Low quality primers.

Poor TA cloning efficiency

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Too much enzyme.
PCR products have blunt
ends.

Change from 3-step cycling to 2-step cycling.
Decrease the number of cycles by 2–5 cycles.
Reduce the amount of template DNA.
Use fresh primers.
Decrease the primer concentration step-wise from
0.3 µM to 0.1 µM.
Redesign primers.
(Longer primers may eliminate smearing or extra
bands.)
Reduce enzyme to 0.5–0.8U/50 µL reaction
Clone the PCR products according to general bluntend cloning guidelines.
Use TArget Clone™ -Plus- (Code No. TAK-201)[see related products].

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www.toyobo.co.jp/e/bio(2) Multiplex PCR

Symptom

Cause

Solution

Cycling conditions are notsuitable.

Increase the extension time up to 60 s/kb.
Increase the number of cycles by 2–5 cycles.
Use a 3-step cycle instead of a 2-step cycle.
Lower annealing temperature in the 3-step cycle
step-wise up to Tm−5°C.
Increase the amount of template DNA.
Change the DNA purification method.
Use purified templates.
Decrease the amount of cDNA to reduce inhibition
by RNA contaminants.
Degrade or eliminate RNA in the cDNA sample.
Use fresh primers.
Redesign primers.
Increase enzyme concentration to 1.5–2.0 U/50 µL.

Template DNA is of low
quality and/or quantity.
No PCR product/low yield

Poor primer performance.

Amplification bias
between targets

Smearing/Extra bands

Enzyme concentration is
too low.
Cycling conditions are notsuitable.
The concentration ofprimers is inappropriate.

Poor primer performance.
Cycling conditions are notsuitable.
Too much template DNA.
Poor primer performance.

Low quality primers.
Too much enzyme.

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Lower annealing temperature in 3-step cycling
step-wise up to Tm−5°C.
Increase the concentration of primers for the targets
exhibiting faint bands, or decrease theconcentration of primers for the targets exhibiting
excessive amplification.
Decrease the difference in Tm between primers.
Change from 3-step cycling to 2-step cycling.
Decrease the number of cycles by 2–5 cycles.
Reduce the amount of template DNA.
Use fresh primers.
Decrease the primer concentration step-wise from
0.3 µM to 0.1 µM.
Redesign primers.
Reduce enzyme to 0.5–0.8U/50 µL reaction.

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www.toyobo.co.jp/e/bio(3) PCR using bisulfite-treated DNA

Symptom

Cause

Solution

Cycling conditions are notsuitable.

Increase the extension time up to 60 s/kb.
Increase the number of cycles by 2–5 cycles.
Use a 3-step cycle instead of a 2-step cycle.
Lower annealing temperature in the 3-step cycle
step-wise up to Tm−5°C.
Increase the amount of template DNA.
Change the DNA purification method.
Use purified templates.
Decrease the amount of cDNA to reduce inhibition
by RNA contaminants.
Degrade or eliminate RNA in the DNA sample.
Use fresh primers.
Redesign primers.
Increase enzyme concentration to 1.5–2.0 U/50 µL.

Template DNA is of low
quality and/or quantity.
No PCR product/low yield

Poor primer performance.

Smearing/Extra bands

Conversion efficiency is low
Poor TA cloning efficiency

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Enzyme concentration is
too low.
Cycling conditions are notsuitable.
Too much template DNA.
Poor primer performance.

Low quality primers.
Too much enzyme
Low bisulfite treatment
efficiency.
PCR products have blunt
ends.

Increase the annealing temperature up to Tm+5°C.
Decrease the number of cycles from 40 to ~30–35.
Reduce the amount of template DNA.
Use fresh primers.
Decrease the primer concentrations step-wise from
0.3 µM to 0.1 µM.
Redesign primers.
Reduce enzyme to 0.5–0.8U/ 50µl reaction.
Confirm the conversion efficiency using artificiallymethylated DNA as a positive control.
Clone the PCR products according to general bluntend cloning guidelines.
Use TArget Clone™ -Plus- (Code No. TAK-201)[see related products].

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

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

3)

Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126:
762-8 (1999)[9] Related roducts
Product name
TArget Clone -Plus10× A-attachment mix
Ligation high Ver.2

No. ofReactions
10 reactions
25 reactions
750 µL(100 reactions)

Code No.
TAK-201
TAK-301
LGK-201

TArget Clone -Plus- is a highly efficient TA cloning kit. The kit can be applied to the TA
cloning of blunt-ended PCR products amplified using KOD -Plus- [Code No. KOD-201],
KOD -Plus- Neo [Code No. KOD-401], KOD FX [Code No. KFX-101], KOD FX Neo[Code No. KFX-201] or KOD -Multi & Epi-TM [Code No. KME-101]. The kit contains the
pTA2 Vector, 2× Ligation Buffer, T4 DNA Ligase, and 10× A-attachment Mix.
The 10× A-attachment mix is a reagent comprising anti-KOD DNA polymerase antibody
that specifically inhibits KOD 3ʹ5ʹ 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], KOD -Plus- Neo [Code No. KOD-401], KOD FX[Code No. KFX-101] and KOD FX Neo [Code No. KFX-201] all possess blunt ends dueto the 3ʹ5ʹ exonuclease activity of the KOD DNA polymerase. The 10× A-attachment
mix allows for PCR products to acquire overhanging dA at the 3ʹ-ends.
Products with 3ʹ-dA overhangs can be directly cloned into various T-vectors using ligation
reagents such as Ligation high Ver.2 [Code No. LGK-201].

Principle of the 10× A-attachment mix:

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

CHINA
TOYOBO (SHANHAI) BIOTECH, CO., LTD.
Tel(86)-21-58794900.4140

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.