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Instruction manual for KOD -Multi & Epi- 2004 F1440K

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

CAUTION
All reagents in this kit are intended for research purposes only. Do not use for diagnostics or clinical purposes. Please observe general
laboratory precautions and safety measures while using this kit.

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[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 and
capillary 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.

- 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 an
advantage (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 long
targets.)

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[2] Components KOD -Multi & Epi-TM (1.0 U/µL)* 200 µL × 1

2× PCR Buffer for KOD -Multi & Epi-TM** 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 5ʹ-
and 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-end
of 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 for
many 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, 0.5
μM.

(2) Multiplex PCR

- 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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(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 that
allows 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 (50
µL reaction):

Sample type Appropriate amount Typical amount

Genomic DNA: Eukaryotic 5–200 ng 50 ng
Genomic DNA: Prokaryotic 0.1–100 ng 10 ng
Plasmid DNA 10 pg–50 ng 1 ng
cDNA ~200 ng (RNA equiv.) a 50 ng (RNA equiv.)
Bisulfite-treated DNAb ~200 ng
Crude sample (blood, lysates, etc.)c

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.
c
Various 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 be
cloned 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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[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 Final Concentration

PCR-grade water X μL
2× PCR buffer 25 μL 1×
for KOD -Multi Epi-TM*

10 pmol/μL Primer #1 1.5 μL 0.3 μM

10 pmol/μL Primer #2 1.5 μL 0.3 μM

Template DNA Y μL Genomic DNA ≤ 200 ng/50 μL

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

KOD -Multi & Epi-TM 1 μL 1.0 U/50 μL

Total reaction volume 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 be
increased 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 2-
step 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 30 cyclesc
b
Extension: 68°C, X s

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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
30 cyclesc
Extension: 68°C, X sb

Notes:

aThe annealing temperature can be decreased in decrements of up to Tm−5°C to enhance

amplification yield.
bThe extension time should be determined according to the following table.

Purified DNA (Genomic DNA, Plasmid Crude sample

DNA, cDNA) (Tissue, lysate),
Target does not Target
contain GC contains GC
cluster* cluster*
Extension time 15–30 s/kb# 30 s/kb 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 the
extension 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 Final Concentration

PCR-grade water X µL
2× PCR buffer 25 µL 1×
for KOD -Multi & Epi-TM*
Primers Y µL 0.3 µM each#
Genomic DNA ≤ 200 ng/50 µL
Z µL Plasmid DNA ≤5 0 ng/50 µL
Template DNA
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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#
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-specific
or smeared bands are observed.
- Amplification bias in multiplex PCR can be improved by increasing the concentration of
primers that yield relatively weak bands, or by decreasing the concentration of primers that
exhibit 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 2-
step 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 25 cyclesc
Extension: 68°C, X sb

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

Pre-denaturation : 94°C, 2 min

Denaturation : 98°C, 10 s
25 cyclesc
Extension : 68°C, X sb

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 or
smeared 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 > 10
<Purified DNA, cDNA> Extension times
3-step cycle 15–30 s 30–60 s 30 s/kb* 30–60 s/kb*
*
2-step cycle 30 s 30–60 s 30 s/kb 30–60 s/kb*
<Crude samples> Extension times
3- and 2-step cycles 60 s 60 s 60 s/kb* 60 s/kb*
*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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(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 Sequence Primer length T m (℃) GC(%) Target size

Chrome9_1 kb_F primer GAATTCCATATCTTTGCCAAACACTTGGTG 30 72.1 40.0
1 kb
Chrome9_1 kb_R primer CCATGGGAAATGTGTTGAAGAAAACAAAGTG 31 73.5 38.7
MSH6_1.8 kb_F primer CAGAAGAGGAAGAAGAGATGGAGGT 25 65.9 48.0
1.8 kb
MSH6_1.8 kb_R primer GGAGGTAAGAAGAGACAGGCAAAGT 25 65.7 48.0
BRCA2_2.3 kb_F primer CAGGTCTTAACCTAGCAGAGGAGGT 25 65.7 52.0
2.3 kb
BRCA2_2.3 kb_R primer GGTTGGTCTGCCTGTAGTAATCAAG 25 65.5 48.0
WT-1_2.5 kb_F primer GAGGTCGAGCCACTCTTTATTACG 24 65.7 50.0
2.5 kb
WT-1_2.5 kb_R primer TCTGACTCCCTTCGTCTAGTCTCTG 25 66.0 52.0
FANCE_3 kb_F primer CAGTCTTCCGTTAGATATCCTGAGC 25 64.9 48.0
3 kb
FANCE_3 kb_R primer CTTCTGCCTAGATCTCCAGAGGATT 25 65.8 48.0
RAD51D_4 kb_F primer ACAGTGAGACGTGAGACCCTATCTC 25 65.7 52.0
4 kb
RAD51D_4 kb_R primer CACAAATCTATTGCCCTGATAGCAT 25 65.7 40.0
KRAS_5 kb_F primer CTTCCTGTGGGCTAGAGATACACTG 25 65.9 52.0
5 kb
KRAS_5 kb_R primer CAAGCAACTAAGGTGAGTGGAAGAG 25 66.0 48.0
BRCA1_7 kb_F primer GCCCTTTAAGCAAAGACAGTAGTCC 25 65.8 48.0
7 kb
BRCA1_7 kb_R primer CATCTCTGTCTGGTCAATCCCTTAC 25 65.7 48.0
DDB2_10 kb_F primer GCAGCAATAGTGGAAGACTGGTTAC 25 65.7 48.0
10 kb
DDB2_10 kb_R primer GCTAGAGCCACCATTAGACTCAGAC 25 65.2 52.0

3. PCR using bisulfite-treated DNA

(1) Reaction set-up

Targets up to 1.5 kb can be amplified using bisulfite-treated DNA.

Component Volume Final Concentration

PCR grade water X µL
2× PCR buffer 25 µL 1×
for KOD -Multi & Epi-TM*
Primers Y 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

*PCR buffer for KOD -Multi & Epi-TM contains dNTPs (dATP, dGTP dCTP, dTTP), and 2 mM
Mg2+.
#
The primer concentrations should be reduced step-wise in decrements of up to 0.1 mM (final).

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(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 40 cyclesc
Extension: 68°C, X sb

Notes:

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

< 500 bp 500–1,000 bp ≥ 1,000 bp

Extension time 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】
M 1 2 3 4 M
0.3 µM each M. 100 bp DNA ladder
【Sample】 1. TGFβ 917 bp (AT content*1 : 69.9%)
55 ng (bisulfite-treated DNA) 1kb 2. BRCA2 1134 bp (AT content*1 : 63.9%)
/ 50 µl reaction
500bp 3. APOE 1487bp (AT content*1 : 69.1%)
【PCR cycle】
4. TGFβ 1583 bp (AT content*1 : 61.8%)
94℃, 2 min.
98℃, 10 sec.
60℃, 30 sec. 40 cycles 100bp
68℃, 30 sec./ kb
4℃, hold *1 The AT content after bisulfite treatment are shown.

Primer Sequence Primer length T m (℃) GC(%) Target size

TGFβ_917 bp_F TGGATTTTAAAGTTTTAGTTTTTTTTAGG 29 61.6 20.7
917 bp
TGFβ_917 bp_R CAACTACTTCCAACCTCCCATAATA 25 62.8 40.0
BRCA2_1134 bp_F GGGGAATAGGTTTTGAGAGAATATT 25 62.2 36.0
1134 bp
BRCA2_1134 bp_R ATACCACTAACCACATTAAACACTC 25 58.8 36.0
APOE_1487 bp_F GGTTTTTTTAAGTAGGGTGGTTTG 24 62.3 37.5
1487 bp
APOE_1487 bp_R TCAAAAAACCAATTTCCTCTTTATC 25 62.0 28.0
TGFβ_1583 bp_F TTGGAGAAAGTTGATTTAGAGTTTG 25 61.3 32.0
1583 bp
TGFβ_1583 bp_R CAACTACTTCCAACCTCCCATAATA 25 62.8 40.0

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[7] Troubleshooting

(1) Singleplex PCR

Symptom Cause Solution

Cycling conditions are not Increase the extension time up to 60 s/kb.
suitable. 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.
Template DNA is of low Increase the amount of template DNA.
quality and/or quantity. Change the DNA purification method.
Use purified templates.
Decrease the amount of cDNA to reduce inhibition
No PCR product/low yield
by RNA contaminants.
Degrade or eliminate RNA in the cDNA sample.
Poor primer performance. 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 3.0
µM in cases where degenerate primers are being
used.
Use fresh primers.
Redesign primers.
Enzyme concentration is Increase enzyme concentration to 1.5–2.0 U/50 µL.
too low.
Cycling conditions are not Change from 3-step cycling to 2-step cycling.
suitable. Decrease the number of cycles by 2–5 cycles.
Too much template DNA. Reduce the amount of template DNA.
Poor primer performance. Use fresh primers.
Decrease the primer concentration step-wise from
Smearing/Extra bands
0.3 µM to 0.1 µM.
Low quality primers. Redesign primers.
(Longer primers may eliminate smearing or extra
bands.)
Too much enzyme. Reduce enzyme to 0.5–0.8U/50 µL reaction
Poor TA cloning efficiency PCR products have blunt Clone the PCR products according to general blunt-
ends. end cloning guidelines.
Use TArget Clone™ -Plus- (Code No. TAK-201)
[see related products].

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(2) Multiplex PCR

Symptom Cause Solution

Cycling conditions are not Increase the extension time up to 60 s/kb.
suitable. 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.
Template DNA is of low Increase the amount of template DNA.
quality and/or quantity. Change the DNA purification method.
No PCR product/low yield Use purified templates.
Decrease the amount of cDNA to reduce inhibition
by RNA contaminants.
Degrade or eliminate RNA in the cDNA sample.
Poor primer performance. Use fresh primers.
Redesign primers.
Enzyme concentration is Increase enzyme concentration to 1.5–2.0 U/50 µL.
too low.
Cycling conditions are not Lower annealing temperature in 3-step cycling
suitable. step-wise up to Tm−5°C.
The concentration of Increase the concentration of primers for the targets
Amplification bias
primers is inappropriate. exhibiting faint bands, or decrease the
between targets
concentration of primers for the targets exhibiting
excessive amplification.
Poor primer performance. Decrease the difference in Tm between primers.
Cycling conditions are not Change from 3-step cycling to 2-step cycling.
suitable. Decrease the number of cycles by 2–5 cycles.
Too much template DNA. Reduce the amount of template DNA.
Poor primer performance. Use fresh primers.
Smearing/Extra bands
Decrease the primer concentration step-wise from
0.3 µM to 0.1 µM.
Low quality primers. Redesign primers.
Too much enzyme. Reduce enzyme to 0.5–0.8U/50 µL reaction.

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(3) PCR using bisulfite-treated DNA

Symptom Cause Solution

Cycling conditions are not Increase the extension time up to 60 s/kb.
suitable. 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.
Template DNA is of low Increase the amount of template DNA.
quality and/or quantity. Change the DNA purification method.
No PCR product/low yield Use purified templates.
Decrease the amount of cDNA to reduce inhibition
by RNA contaminants.
Degrade or eliminate RNA in the DNA sample.
Poor primer performance. Use fresh primers.
Redesign primers.
Enzyme concentration is Increase enzyme concentration to 1.5–2.0 U/50 µL.
too low.
Cycling conditions are not Increase the annealing temperature up to Tm+5°C.
suitable. Decrease the number of cycles from 40 to ~30–35.
Too much template DNA. Reduce the amount of template DNA.
Poor primer performance. Use fresh primers.
Smearing/Extra bands
Decrease the primer concentrations step-wise from
0.3 µM to 0.1 µM.
Low quality primers. Redesign primers.
Too much enzyme Reduce enzyme to 0.5–0.8U/ 50µl reaction.
Conversion efficiency is low Low bisulfite treatment Confirm the conversion efficiency using artificially
efficiency. methylated DNA as a positive control.
Poor TA cloning efficiency PCR products have blunt Clone the PCR products according to general blunt-
ends. end cloning guidelines.
Use TArget Clone™ -Plus- (Code No. TAK-201)
[see related products].

JAPAN CHINA
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[ 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 No. of Code No.
Reactions
TArget Clone -Plus- 10 reactions TAK-201
10× A-attachment mix 25 reactions TAK-301
Ligation high Ver.2 750 µL LGK-201
(100 reactions)

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 due
to 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:

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