Raw Material Catalog

Real-time PCR Master Mix
Raw Materials
PCR Kits
NGS Related Products

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1 Real-time PCR Master Mix
Real-time PCR selection guide1

Real-time PCR Master Mix series

2

THUNDERBIRDTM qPCR Mix series

4

KOD SYBR qPCR Mix

6

THUNDERBIRD

8

®

TM

Probe One-step qRT-PCR Mix

10

RNA-directTM Realtime PCR Master Mix series

Materials
2 Raw
rTaq DNA Polymerase Hot Start / rTaq DNA Polymerase Hot Start <Glycerol Free>12

rTaq DNA Polymerase

12

10x Buffer for Taq Hot Start

13

Raw Material related Tth/TTx DNA Polymerase

14

Hot Start TTx DNA Polymerase / Hot Start TTx DNA Polymerase <Glycerol Free>15

Hot Start rTth DNA Polymerase

15

rTth DNA Polymerase

16

2x Buffer for rTth/TTx (DNA)16

5x Buffer for rTth/TTx (DNA/RNA) 5x Buffer for rTth/TTx (DNA/RNA) <Glycerol Free>

1650mM Mn(OAc)21725mM MgCl2

17

50x ROX reference dye

17anti-Taq high18

Anti-Taq DNA Polymerase Antibody 1,2 <Glycerol Free>18

anti-Taq Neo

18

anti-Taq Neo <Glycerol Free>

18

RNase Inhibitor, Recombinant19

Uracil-DNA Glycosylase, Heat-labile

19

Uracil-DNA Glycosylase, Heat-labile <Glycerol Free>

19

ReverTra Ace

20

dNTP (dATP, dCTP, dGTP, dTTP, dUTP)

20

dNTPs Mixture

20

dNTPs Set

20

Thermo T7 RNA Polymerase

21

TM

PCR Kits
3 KOD
One PCR Master Mix

22

KOD One PCR Master Mix -Blue-

22

KOD FX

25

Related Products
4 NGS
GenNext NGS Library Prep Kit

27

TM

28

GenNext NGS Library Quanti ication Kit
TM

http://www.bio-toyobo.cn/

Real-time PCR Master Mix selection guide
TOYOBO has various realtime PCR master mixes from which to choose. The following table shows the characteristics ofeach product. The Realtime PCR Master Mix series and the THUNDERBIRDTM qPCR Mix series are Taq DNA
polymerase-based 2x master mixes for rea-ltime PCR, which contain all of the necessary reaction components, except forthe primer and probe. The THUNDERBIRDTM series shows reduced primer–dimer formation and ROX dye is provided
separately to enable appropriate application depending on the realtime cycler used. The RNA-directTM series is a 2x
master mix for one-step realtime RT-PCR using a thermostable DNA polymerase derived from Thermus thermophilus(Tth) HB8. Tth DNA polymerase exhibits reverse transcriptase activity in the presence of Mn2+ ions. This system allows
one-step realtime RT-PCR, including both reverse transcription and PCR steps. THUNDERBIRDTM Probe One-step
qRT-PCR Kit was developed with a focus on the sensitivity as a one-step real-time RT-PCR kit using the highly ef icient
reverse transcriptase “ReverTra AceTM” and Tth DNA Polymerase as a PCR enzyme.
KOD SYBR® qPCR Mix contains a 3′ 5′ exonuclease-de icient KOD DNA Polymerase and an optimized buffer. The reagent
can also be used for the ampli ication of long (<2 kb) and GC-rich targets. It is also applicable to ampli ication from crude
samples, such as whole blood, microorganisms, and various lysates from animal and plant tissues.

Passivereference

Sensitivity

Specificity

Efficiency

Long target
amplification

GC-rich targets

Amplificationfrom crude
samples

polymerase





+++

++

+++

++

+

+

SYBR® Green Realtime PCR Master Mix

SYBR®





+++

++

+++

++

+

+

THUNDERBIRDTM Probe qPCR Mix

Probe



*

+++

+++

++

+

+

+

+

+

+

+++

+++

+++

++

++

Referencepage

Hot start(Antibody)rTaq DNA

One-step
RT-PCR

Probe

Assay type

Template

Realtime PCR Master Mix

Enzyme Product Name

2

DNA

4
THUNDERBIRDTM SYBR® qPCR Mix

SYBR

®



*

+++

+++

++

KOD SYBR® qPCR Mix

SYBR®



*

+++

+++

+++

RNA-direct™
Realtime PCR Master Mix

Probe





++

++

++

KOD
exo(-) DNA
polymerase

rTth DNA
polymerase(<2kb)++

RNA

1enzymeRNA-direct™
SYBR® Green Realtime PCR Master Mix

SYBR

THUNDERBIRDTM
Probe One-step qRT-PCR Kit

Probe



®

10




++

++

++

++

++

++



*

+++

+++

+++

++

++

+++

1enzyme
2enzymes**+++: Best, ++: Excellent, +: Good,  : Applicable*50x ROX reference dye is individually supplied.
**This product contains ReverTra AceTM and Tth DNA Polymerase.

Effective
qPCR analysis
High efficient assay using
standard conditions！

TaqMan® assay

Suitable for difficult Targets &！
Samples
Amplification of GC-rich targets
Amplification of long targets up to 2 kb
Amplification from crude samples

SYBR® Green I assay

SYBR® Green I assay

Realtime PCR series
THUNDERBIRDTM series*
Realtime PCR Master Mix
TM

THUNDERBIRD Probe qPCR Mix

KOD SYBR® qPCR Mix*SYBR® Green Realtime PCR Master Mix
TM

6

®

THUNDERBIRD SYBR qPCR Mix*50x ROX reference dye is individually supplied to enable appropriate application
depending on the real-time cycler used.

1

http://www.bio-toyobo.cn/

8High efficient real-time PCR Master Mix

Real-time PCR Reagents

Realtime PCR Master Mix SeriesStore at -20 °C
Components:<Probe Version>
Realtime PCR Master Mix*
5 x 1 ml (Code No. QPK-101)(Code No. QPK-119CH)
50 ml
100 ml (Code No. QPK-159)

Realtime PCR Master Mix Series is a Taq DNA
polymerase-based 2x master mix for real-time PCR, whichcontains all components, except for the primer and probe.
Realtime PCR Master Mix is applicable in TaqMan® assays or
hybridization probe assays, in combination with each probe.
SYBR® Green Realtime PCR Master Mix is applicable forintercalation assay with SYBR® Green I.

®

<SYBR Green Version>
SYBR® Green Realtime PCR Master Mix*
5 x 1 ml (Code No. QPK-201)
100 ml (Code No. QPK-259)*The reagents contain 2x ROX reference dye.

This product can be applied to one-step RT-PCR by adding
reverse transcriptase (ReverTra AceTM).

Features
High specificity and effective amplification enable the detection of low-copy targets.
Table

Compatible real-time PCR instruments
ABI PRISM® 7000
ABI PRISM® 7700
Applied Biosystems® 7300
Applied Biosystems® 7900HT
Applied Biosystems® StepOne
Applied Biosystems® StepOne Plus

Applied
Biosystems

Roche
Diagnostics

LightCycler® 1.x / 2.0
LightCycler® Nano
LightCycler® 480 / LightCycler® 96

Bio-Rad/MJ

MiniOpticon / CFX96 Touch

TaKaRaThermal Cycler Dice® Real Time System

QIAGEN

Rotor-Gene

TM

TM

TM

TM

Application data
1. Detection of -actin gene
Amplification of the -actin gene was carried out using serially diluted genomic DNA solutions (10n dilutions; 30 ng-3mg) with real-time PCR kits for the SYBR® Green assay. SYBR® Green Realtime PCR Master Mix [Code No. QPK-201]
showed greater sensitivity and efficiency than other kits (company A ). Moreover, SYBR® Green Realtime PCR MasterMix generated fewer primer dimers than the other kits.
SYBR® Green
Realtime PCR Master Mix

Amplification curve

Company A

Melting curve

Amplification curve

Melting curve

2. Detection of CYP2C19*2 SNP
A single nucleotide polymorphism (SNP) of CYP2C19*2 detected using human genomic DNA with allele specific
TaqMan® probes and Realtime PCR Master Mix [Code No. QPK-101] .
Fwd primer

A/ A

Rev primer

G/ G

T
A

Allele A

Fwd primer

Allele G

TaqMan Probe

TaqMan Probe

Rev primer

G
C
Green: A specific probe
Blue: G specific probe2

http://www.bio-toyobo.cn/

A/ GHigh efficient real-time PCR Master Mix

The sensitivity and quantitativity of two master mix reagents were compared by detecting serially (3n) diluted HPV DNA(100, 33, 11 copies) using TaqMan® probe. Real-time PCR Master Mix (TOYOBO) exhibited higher sensitivity and PCRefficiency.
Real-time PCR Master Mix (TOYOBO)

Company A

100 copies

100 copies

11 copies

11 copies

4. Detection of Enteric bacteria DNA
The target genes were detected from serially (4n) diluted purified genomic DNA of Salmonella, Escherichia coli (VTEC)
and Shigella cells by triplex detection systems with TaqMan® probes labeled by different fluorescent dyes. No
significant differences of PCR efficiency and correlation coefficient were observed between the triplex and singleplex
systems.
MulƟplex
Salmonella

E. Coli (VTEC)

Shigella

40.00

40.00

35.00

35.00

35.00

30.00

30.00

30.00

25.00

25.00

y = -3.639x + 41.248
R² = 0.9909

20.00

0

1

2
Copies (log)20.00

Single

4

30.00

0

1

2
Copies (log)

Ct
20.00

Single

15.00
3

4

0

1

2
Copies (log)y = -3.639x + 41.248
R² = 0.9909
y = -3.6953x + 37.107
R² = 0.9959
y = -3.4663x + 39.207
R² = 0.9925

25.00

y = -3.4663x + 39.207
R² = 0.9925
y = -3.3858x + 39.273
R² = 0.9962

MulƟ

y = -3.409x + 36.247
R² = 0.9981

15.00
3

35.00

25.00

y = -3.6953x + 37.107
R² = 0.9959

MulƟy = -3.4448x + 41.194
R² = 0.9801

15.00

40.00

Ct

Ct

40.00

Ct

Real-time PCR Reagents

3. Detection of papilloma virus (HPV) DNA

MulƟSingle

3

20.00
4

15.00
0

1

Salmonella
VT
Shigella2
Copies (log)34Related products
Realtime PCR Master Mix (Code No. QPK-101) is standard type of master mix based on Taq DNA Polymerase. It consists ofvarious components such as enzymes, buffers, dNTPs etc. TOYOBO supplies basic and optional components so that
researchers can modify the reagents depending on the situation. By using the standard option, the product can bereconstituted.Some of the components (enzymes or buffers) contain glycerol which inhibit lyophilization. TOYOBO supply
glycerol-free components.
Options

10x Buffer for Taq Hot Start









dNTPs







50x ROX reference dye

Uracil-DNA-Glycosylase



dUTP





12





13







20







17
19



20


ReverTra AceTM
Glycerol freecomponent

rTaq DNA Polymerase Hot Start<Glycerol Free>

Uracil-DNA-Glycosylase<Glycerol Free>

3

Reference pageOne-step RT-PCR

carry-over prevention& Glycerol freecarry-over prevention

rTaq DNA Polymerase Hot Start

Glycerol free

Without ROX dye

Optional
componentStandard

Basic
component

Product name

20



12



19

http://www.bio-toyobo.cn/

HighHighefficientefficientreal-timereal-timePCR
PCRMaster
MasterMix
Mix
TM

THUNDERBIRD qPCR Mix SeriesStore at -20 °C
Components:®

Code No. QPS-101（TaqMan probe version）
200 reactions [50 l per reaction]

THUNDERBIRTM Probe qPCR Mix
50x ROX reference dye

THUNDERBIRD Probe and SYBR qPCR Mix is a Taq DNA
polymerase-based highly ef icient 2x master mix for®
®
real-time PCR using TaqMan probes and SYBR Green I.
The master mix contains all required components, except
for ROX reference dye, probe and primers (50x ROX
reference dye is individually supplied with this kit). The
master mix facilitates reaction setup, and improves the
reproducibility of experiments.
These products are improved versions of Realtime PCR®
Master Mix (Code No. QPK-101) and SYBR Green
Realtime PCR Master Mix (Code No. QPK-201) . In
particular, reaction speci icity is enhanced.
TM

3 x 1.67 ml
250 l®

Code No. QPS-201 (SYBR Green version)®

200 reactions [50 l per reaction]

THUNDERBIRDTM SYBR® qPCR Mix
50x ROX reference dye

3 x 1.67 ml
250 lFeatures
The specificity for the detection of low-copy targets is improved.
The dispersion of PCR efficiency between targets is reduced by a new PCR enhancer*. (*Patent pending)High specificity and effective amplification enable the detection of a broad dynamic range.
Applicable to most real-time cyclers (i.e. block type and glass capillary type).
THUNDERBIRDTM SYBR® qPCR Mix
Melting curve

Amplification curve
10-1

Company A
Primerdimer

Company B
Primer
dimer

10-1

10-1
10-5

10-5

10-5dilution
0

5

10

15

20

25 30

Cycles35 40 45

0

5

10

15

20

25 30

35 40 45

0

5

10

15

20

25 30

CyclesCycles35 40

-actin gene was detectedwith serially diluted cDNAfrom HeLa cell total RNA.
THUNDERBIRDTM SYBR®
qPCR Mix [Code No.
QPS-201] showed greater
sensitivity and efficiency than
45 other kits (companies A and
B).

Fig. Comparison of the sensitivity and efficiency of the SYBR® Green I assay

Applications
Real-time PCR
Table

Compatible real-time PCR instruments

Applied
Biosystems

ABI PRISM® 7000
ABI PRISM® 7700
Applied Biosystems® 7300
Applied Biosystems® 7500/7500Fast
Applied Biosystems® 7900HT
Applied Biosystems® StepOne
Applied Biosystems® StepOne Plus
ViiATM 7 QuantStudio®
TM

TM

Roche
Diagnostics

LightCycler® 1.x / 2.0
LightCycler® Nano
LightCycler® 480 / LightCycler® 96

Bio-Rad/MJ

MiniOpticon / CFX96 Touch

Agilent Technologies

Mx3000P/ Mx3005P/ Mx4000

TaKaRaThermal Cycler Dice® Real Time System

QIAGEN

Rotor-Gene

4

TM

TM

http://www.bio-toyobo.cn/

High efficient real-time PCR Master Mix

Application data
1. Comparison of the PCR efficiency
Norovirus G1 and human GAPDH genes were detected using serially diluted cDNA samples by SYBR® Green I and
TaqMan® realtime PCR. THUNDERBIRDTM qPCR Master Mix showed greater efficiency than other reagents.

10 0 dilution

THUNDERBIRDTM
qPCR Mix

10 0 dilution

Company A
Company B

10 -4
10 -4
Detection of Norovirus G1 gene
by SYBR® Green I assay.
[Roche Diagnostics LightCycler 1.1]

Detection of human GAPDH geneby TaqMan® assay.
[Roche Diagnostics LightCycler 1.1]

2. Verification of the measurement accuracy
Human GAPDH genes were detected using serially 2.5 fold diluted cDNA synthesized from HeLa cell total RNA by
SYBR® Green I assay (n=4). THUNDERBIRDTM SYBR® qPCR Mix successfully detected the differences between
dilutions.
1.000 E+1

24.5

Slope: -3.2587
Correlation factor: 0.9956

24.0

⊿Rn

Ct Value

23.5
23.0
22.5
22.0
21.5

1.000

21.0
20.5
-1.00

-0.90

-0.80

-0.70

-0.60

-0.50

-0.40

-0.30

-0.20

-0.10

0.00

Log (Template concentration)

1.000 E-1

0

5

10

15

20
Cycles

25

30

35

®

Detection of human GAPDH gene by SYBR Green I assay.
[Applied Biosystems 7900HT]5

http://www.bio-toyobo.cn/

High efficient real-time PCR Master Mix

KOD SYBR® qPCR Mix

CHigh G

Crude

Long

Store at -20 °C
Components:

Code No. QKD-201
200 reactions [50 l per reaction]®

3 x 1.67 ml
250 l

KOD SYBR qPCR Mix
50x ROX reference dye

KOD SYBR qPCR Mix is a highly ef icient 2x master mix for®
real-time PCR using SYBR Green I and the 3’5’ exonucleasede icient KOD DNA Polymerase. The master mix contains allof the required components, except for the primers and the
ROX reference dye (50x ROX reference dye is supplied
separately with this kit). The master mix was developed
based on the unique properties (high ef iciency, robustness)of KOD DNA Polymerase to enhance the convenience andversatility of the SYBR® Green I assay.
®

Table Comparison of properties with the conventional masterFeatures

Conventional(Taq based)

KOD SYBR® qPCR

EnzymeTaq DNA

KOD DNA Polymerase[exo(-) mutant]

Amplification
size

70 ~ 150 bp(Maximum: 300 bp)

70 bp ~ 2 kb

High GC
Targets

Susceptible

Not susceptible

Inhibition by
impurities incrude samples

Susceptible

Not susceptible(Suitable for amplificationfrom cruｄe specimens)

Quantitative amplification for long targets(~ 2kb).
M®

KOD SYBR qPCR Mix

Same primers

Conventional master mix（Taq based)

Amplification curve104

1.9 kb104
100copy

Target: TGFβ (1,918 bp)
Template: genomic DNA
M: 1 kb Ladder

100copy

Melting curve<Electrophoresis analysis>Fig. Long target amplification [ABI StepOnePlusTM]

Efficient amplification for GC-rich targets
High efficient master mix（Company A）®

KOD SYBR qPCR Mix
Amplification curve

5-1dilution

5-1dilution

5-５dilution

5-５dilution

Melting curve

Target: IGF2R (189 bp / GC content: 83%)
Template: HeLa cDNA was synthesized using
ReverTra AceTM qPCR RT Kit (Code No.FSQ-101)with total RNA from HeLa cells.
Fig. Amplification of GC rich targets [ABI StepOnePlusTM]

Primer dimer

Effective amplification from crude samples.
Effective amplification can be achieved using crude samples, as shown in the table.
This reagent can be used for genotyping or SNP analysis using crude specimens.

Table Applicable samples● Genotyping of knock-in mice using mouse-tail lysates
Wild type (WT)

100 bp / Tm (calculated)： 79°C

Common F Primer
CreER knock-in(KI)

WT Primer

1.200

0.800

CreER

Cre Primer

341 bp / Tm (calculated)： 84°C

Target: Region contains the targeting site (Cre ER)(WT: 100 bp, KI: 341 bp)

Melting curve

1.000

WT/WT

0.600
0.400

K I/K I

whole blood
nail (toe)hair rootoral mucosa
cultured cells
animal tissue
plant tissue

ca. 1% (final)ca. 1mm
1~2 mmsuspension
3~ 10 cells
lysate (p 26)
lysate (p 26)

WT/K I

0.200
0.000-0.200Template: Mouse tail lysate (alkaline lysis method, p 26)
Primer ratio: F: WT: Cre = 0.2: 0.2: 0.67 mM (final)
Sample: Mouse tail lysate 2 l / 20 l reaction

Fig. One-tube mouse genotyping
using melting curve analysis[ABI 7500 Fast]６

hhttp://www.bio-toyobo.cn/

High efficient real-time PCR Master Mix
Applications
Real-time PCR
Table

Compatible real-time PCR instruments

Applied
Biosystems

ABI PRISM® 7000
ABI PRISM® 7700
Applied Biosystems® 7300
Applied Biosystems® 7500/7500Fast
Applied Biosystems® 7900HT
Applied Biosystems® StepOne
Applied Biosystems® StepOnePlus
ViiA7TM QuantaStudio®
TM

Roche
Diagnostics

LightCycler® 1.x / 2.0
LightCycler® Nano
LightCycler® 480 / LightCycler® 96

Bio-Rad/MJ

MiniOpticon / CFX96 Touch

Agilent Technologies

Mx3000P/ Mx3005P/ Mx4000

TaKaRaThermal Cycler Dice® Real Time System

QIAGEN

Rotor-Gene

TM

TM

TM

Application data
1. Comparison of the PCR efficiency on a ChIP target
A promoter region having a typical CpG island was amplified using KOD SYBR® qPCR Mix and a conventional mastermix with Taq DNA Polymerase. A quantitable detection was shown by KOD SYBR® qPCR Mix depending on theconcentration of the template DNA. The conventional qPCR master mix based on Taq DNA Polymerase generated
primer dimers mainly.

Conventional master mix（Taq based)

KOD SYBR® qPCR Mix

CCCGCCCGAGAGAGTGACTCTCACGAGAGCCGCGAGAGTCAGCTTGGCCAATCCGTGCGGTCGGCGGCCGCTCCCTTTATAAGCCGACTCGCCCGGCAGCGCACCGGGTTGCGGAGGGTGGGCCTGGGAGGG
GTGGTGGCCATTTTTTGTCTAACCCTAACTGAGAAGGGCGTAGGC
GCCGTGCTTTTGCTCCCCGCGCGCTGTTTTTCTCGCTGACTT

Amplification curve

104

104

101copy

Target: GC content ： 64%, 219 bp ： Homo sapiens telomerase
RNA (TR) gene, promoter and complete sequence
Template: human genomic DNA
Primer: (from a paper using the ChIP technique） ：Blue: primer sequence

101copy

Melting curve

2. SNP analysis using whole blood samples
SNP analysis was performed with a GC tailed primer from whole blood samples using KOD SYBR® qPCR Mix. All types of SNP weresuccessfully determined by KOD SYBR® qPCR Mix. No signal was detected using the Taq-based conventional master mix (data notshown).

5ʼ
CG
GC
CC
Target size: 57bp
Tm of amplicon: 78°C (predicted)
3’<GC tail>Common primer

G specific primer

5’

SNP siteCommon primer
5’

C

CC

CG

3’ A C G
TGA

Mismatch site

C
GT

1.200

G/A
G/G

0.800

ΔTm : >3°C (optimally > 5°C)

Whole blood 2 ｌA specific primer
3’ A T A
TAA

A/A

1.000

0.600
0.400

5’

Target size: 45 bp
3’ Tm of amplicon: 72°C (predicted)

Suspend in 48 ｌ DW
5 ｌ
20 ｌ reaction

0.200
0.000
-0.20060 65 70 75 80 85 90 95

Temperature(℃)

Fig. One-tube ASP-PCR analysis using whole blood
specimen. [ABI 7500 Fast real-time PCR system]7

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High efficient real-time qRT-PCR Kit

THUNDERBIRDTM Probe One-step qRT-PCR Kit
Store at -20 °C
Components:
Code No. QRZ-101
100 reactions [50 μl per reaction]

2x Reaction Buffer*
DNA Polymerase
RT Enzyme Mix
50x ROX reference dye
RNase free water

THUNDERBIRDTM Probe One-step qRT-PCR Kit is a
one-step realtime reverse-transcription polymerase
chain reaction (RT-PCR) kit using the highly ef icient
reverse transcriptase “ReverTra AceTM” and Tth DNA
polymerase as a PCR enzyme. This product can beused mainly in TaqMan® probe assays. The one-stepsystem is suitable for high-throughput analysis
because of its simple reaction setup. In addition, thissystem can reduce the risk of cross-contamination.
The combination of the two enzymes and optimized
buffer system enable the effective detection andquanti ication of a small amount of RNA. This kit canalso detect various kinds of RNA with different
sequences because it is tolerant of target sequence
diversity.

2 × 1.25 ml
125 μl125 μl100 μl2 × 1.25 ml

Code No. QRZ-129B
10,000 reactions [50 μl per reaction]

2x Reaction Buffer*
DNA Polymerase
RT Enzyme Mix

250 ml12.5 ml12.5 ml* 2x Reaction Buffer contains essential components for thereaction (buffer, Mg2+, salts, dATP, dCTP, dGTP, and dUTP, etc.).

Features
Measles virus

Rapid and highly sensitive detection（RNA copy number）
Mumps virus
Hepatitis
C 型肝炎ウイルス
C virus0

Tolerant of target sequence diversity

200

Utilization of dUTP

400

Dengue virus

This kit contains dUTP instead of dTTP in 2× Reaction Buffer.
Therefore, the rate of false-positive detection can be reduced byadding uracil-N-glycosylase (UNG).
*UNG is not supplied with this kit.

800

West Nile
virus

1000

Entero virus

Chikungunya
virus

Tolerant of PCR inhibitors
THUNDERBIRDTM Probe One-step
qRT-PCR Kit

Yellow fever virus

600

Rhino virus
RS virus type A RS virus type B

Company A
THUNDERBIRDTM Probe One-step qRT-PCR Kit
Company A
Company B

Fig The maximum sensitivities of various one-step qRT-PCR kits
A 4n dilution series of various viral RNAs was detected. The primers
and TaqMan® probes were synthesized in accordance with previous
reports. The graph indicates the minimum copy numbers that weredetected by the kits. THUNDERBIRDTM Probe One-step qRT-PCR
Kit was the only kit that detected all viral RNAs tested at highsensitivity (≤30 copies).

+ Hematin+ Hematin

Fig The effect of inhibition by hematin
The addition of 2 mM hematin did not affect the reaction.

Applications
Realtime qRT-PCR
Table

Compatible real-time PCR instruments

Applied
Biosystems

ABI PRISM® 7000
ABI PRISM® 7700
Applied Biosystems® 7300
Applied Biosystems® 7500/7500Fast
Applied Biosystems® 7900HT
Applied Biosystems® StepOne
Applied Biosystems® StepOnePlus
ViiATM QuantaStudio®
TM

TM

8

Roche
Diagnostics

LightCycler® 1.x / 2.0
LightCycler® Nano
LightCycler® 480 / LightCycler® 96

Bio-Rad/MJ

MiniOpticon / CFX96 Touch

Agilent Technologies

Mx3000P/ Mx3005P/ Mx4000

TaKaRaThermal Cycler Dice® Real Time System

QIAGEN

Rotor-Gene

TM

TM

http://www.bio-toyobo.cn/

High efficient real-time qRT-PCR Kit
Application data
1. Comparison of sensitivity of detection of enterovirus RNA
The sensitivity and quantitativity of various kits were compared bydetecting serially (4n ) diluted enterovirus RNA . The primers andprobe were synthesized in accordance with a previous report. Applied
Biosystems® StepOnePlus was used in this experiment.
THUNDERBIRDTM Probe One-step qRT-PCR Kit was the only kit that
detected less than 10 copies of RNA and showed wide-ranging
quantitation. The results indicate that this kit is suitable for the highly
sensitive detection of RNA viruses or mRNA expressed at a low level.

40.0
THUNDERBIRDTM Probe
One-step qRT-PCR Kit

35.0

TM

Ct

Company A

30.0

Company B
Company C

25.0

20.0

0

1

2

3

4

RNA concentration (LOG, 10)

2. Comparison of maximum sensitivity of various kits
The expression levels of IL-1b, TNF-a and GAPDH mRNAs were analyzed using 10n times serially diluted total RNA (1 pg–100ng) by triplex detection systems with TaqMan® probes labeled by different fluorescent dyes (Fig.1). LightCycler® 96 (Roche
Diagnostics) was used in this experiment. HeLa S3 cells were incubated for 20 h after being seeded in six-well plates at 4 × 105
cells/well and treated with or without 100 nM phorbol 12-myristate 13-acetate. Then, the expression levels of mRNA wereanalyzed using purified total RNA from treated cells. The elevations of IL-1b and TNF-a mRNAs were observed upon adding
phorbol 12-myristate 13-acetate (Fig. 2).
No significant differences of PCR efficiency and correlation coefficient were observed between the triplex and singleplex
systems (data not shown).
50.0

40.0

20.0

40.0

TNF-a (PMA treatment - )30.0

TNF-a (PMA treatment + )

Ct

Ct

30.0

IL-1b (PMA treatment - )

20.0
TNF-a
IL-1b
GAPDH

10.0

IL-1b (PMA treatment + )GAPDH (PMA treatment - )

10.0

0.0

GAPDH (PMA treatment + )

0.0
0

2
4
RNA concentration (LOG, 10)

0

6

2

6

4

RNA concentration (LOG, 10)

Fig. 1 Multiplex assay results

Fig. 2 Expression analysis by multiplex assay

3. Simultaneous quantitation of RS virus types A and B from clinical specimens
RT-PCR and qRT-PCR analyses of RS virus were performed using RNA purified from 20 throat swab specimens. In qRT-PCR
with THUNDERBIRDTM Probe One-step qRT-PCR Kit, duplex assay was performed using specific primers and TaqMan® probes
for types A and B in a single tube. LightCycler® 96 (Roche Diagnostics) was used in this experiment. In parallel, an antibody test
was performed using the same swab samples.
The detection of the virus using the THUNDERBIRDTM Probe One-step qRT-PCR Kit showed a high correlation with that by
RT-PCR and successfully gave quantitative values.
Table Results of correlation tests on RS virus detection by three methods.
SpecimennumberAntibody test

RT-PCR (Typing)

#１

+

+ (A)

Quantitative value (Estimated RNA copy number)Type A (FAM, 470nm)
1.6 × 105

Type B (Cy5, 645nm)
-

#２

-

-

-

-

#3

+

+ (A)

8.1 × 104

-

#4

+

+ (A)

2.7 × 105

-

#5

-

+ (A)

9.6 × 102

-

#6

-

+ (A)

3.6 × 103

-

#7

+

+ (A)

-

#8

-

-

2.3 × 106
-

#9

+

+ (A)

1.5 × 105

-

#10

+

+ (A)

-

#11

-

-

6.7 × 105
-

#12

+

+ (A)

1.6 × 105

-

#１3

+

+ (A)

9.4 × 103

-

#14

-

+ (A)

9.5 × 103

#15

+

+ (A)

3.9 × 103

-

#16

+

+ (A)

-

-

2.4 × 105
-

-

#17
#18

-

+ (A)

4.2 × 104

-

#19

-

+ (A)

#20

-

+ (B)

3.4 × 102-

9.6 × 103

９

-

-

-

http://www.bio-toyobo.cn/

One-step real-time RT-PCR Master Mix

RNA-direct™ Realtime PCR Master Mix SeriesStore at -20 °C
Components:<Probe Version>
Code No. QRT-101
100 reactions [50 μl per reaction]

RNA-direct™ Realtime PCR Master Mix*
50 mM Mn(OAc)2

RNA-direct Realtime PCR Master Mix is a 2x master mix for
one-step real-time PCR using a thermostable DNApolymerase derived from Thermus thermophilus (Tth) HB8[p16]. Tth DNA polymerase exhibits reverse transcriptase
activity in the presence of Mn2+ ions. This system allows forone-step real-time PCR, including reverse transcription and
PCR steps. RNA-direct™ Realtime PCR Master Mix is
applicable for TaqMan® assay or hybridization probe assay,
in combination with each probe. RNA-directTM SYBR® Green
Realtime PCR Master Mix can be applied to an intercalation
assay with SYBR® Green I.
TM

Code No. QRT-159
RNA-direct™ Realtime PCR Master Mix*

50 mM Mn(OAc)2

20 ml

®

<SYBR Green Version>
Code No. QRT-201
100 reactions [50 μl per reaction]

RNA-direct™ SYBR® Green
Realtime PCR Master Mix*
50 mM Mn(OAc)2

Suitable for high-throughput real-time PCR and

5 × 0.5 ml
0.5 mlCode No. QRT-259

increases reliability of product, due to lowered risk ofRNA-direct™ SYBR® Green
Realtime PCR Master Mix*

contamination.

Applications

100 mlCode No. QRT-MN1
50 mM Mn(OAc)2

One-step real-time PCR

ABI PRISM® 7000
ABI PRISM® 7700
Applied Biosystems® 7300
Applied Biosystems® 7900HT
Applied Biosystems® StepOne
Applied Biosystems® StepOne Plus

20 ml

*The reagents contain 2x ROX reference dye.

Compatible real-time PCR instruments

Applied
Biosystems

100 mlCode No. QRT-MN1

FeaturesTable

5 × 0.5 ml
0.5 mlRoche
Diagnostics

LightCycler® 1.x / 2.0
LightCycler® Nano
LightCycler® 480 / LightCycler® 96

Bio-Rad/MJ

MiniOpticon / CFX96 Touch

TaKaRaThermal Cycler Dice Real Time System

QIAGEN

Rotor-Gene

TM

TM

TM

TM

®

Application data
1. Detection of hepatitis C virus (HCV)The sensitivity and quantitativity of three one-step qRT-PCR master mix reagents were compared by detecting serially(10n) diluted HCV RNA using TaqMan® probe. RNA-direct™ Real time PCR Master Mix (TOYOBO) exhibited higher
sensitivity and PCR efficiency than other master mixes.
.

106 copies

106 copies

106 copies

102

102

102
Company A

RNA-direct™

Company B

2. Verification of the measurement accuracy

RT

90°C

PCR

95°C

95°C

× 45 cycles

6.0
5.0
4.0

RNA-directTM
SYBR® Green
Realtime PCR Master Mix

3.0
2.0
1.0

Company A

0030sec. 61°C 30sec. 15sec. 55°C
20min.

7.0
Fluorescence(F1)Amplification of G3PDH mRNA was detected using serially diluted poly(A)+ RNA (10n dilution) with SYBR® Green Realtime PCR kits including
Tth DNA polymerase. RNA-direct™ SYBR® Green Realtime PCR MasterMix [Code No. QRT-201)] showed greater sensitivity and signal intensity
than the other kit (company A).

74°C

10

20

30

40

Cycle

Fig. Comparison of the SYBR® Green assay

30sec

15sec (Data collection)
Melting curve
analysis10

http://www.bio-toyobo.cn/

One-step real-time RT-PCR Master Mix
3. Detection of single nucleotide polymorphism (SNP) using fresh whole blood
The SNPs of aldehyde dehydrogenase 2 gene (ALDH2) were analyzed using fresh whole blood samples by RNA-direct™
Realtime PCR Master Mix (QRT-101) and Realtime PCR Master Mix (QPK-101) based on Tth and Taq DNA polymerase,
respectively. QRT-101 successfully detected all types of SNPs (G/G, G/A, A/A) using TaqMan® probe assay. In thisexperiment, the amplification by QRT-101 was performed in the presence of MgCl2 instead of Mn(OAc)2.
DW
RNA-direct™ Realtime PCR Master Mix
25mM MgCl2
10uM Fwd
10uM Rev
10uM Pb1
10uM Pb2
Whole blood

5.4
10
2
0.4
0.4
0.4
0.4
1
20 l

QRT-101 QPK-101
(Tth)
(Taq)
1 2 3 1 2 3

PCR
95°C 95°C

× 50 cycles

1 min. 15sec.

60°C
45sec.
(Data collection)

QRT-101(Tth)

QPK-101（Taq）

Related products
RNA-directTM Realtime PCR Master Mix (Code No. QRT-101) consists of various components such as enzymes, buffers, anddNTPs, etc. TOYOBO supplies components individually contained in RNA-directTM Realtime PCR Master Mix (QRT-101).
Researchers can modify the RT-PCR reaction mixture depending on the situation. By using the standard option, the products(QRT-101) can be reconstituted.Some of the components (enzymes or buffers) contain glycerol which inhibit lyophilization.
TOYOBO supply glycerol-free components.
Options
Tth DNA Polymerase

Reference page













50mM Mn(OAc)2

















17

dNTPs

















20

50x ROX reference dye













17

15

Uracil-DNA-Glycosylase





dUTP






Hot Start TTx DNA Polymerase<Glycerol free>
5x Buffer for rTth/ TTx (DNA/RNA)<Glycerol free>
Uracil-DNA-Glycosylase <Glycerol
free>carry-over prevention& Glycerol freeGlycerol freeHot Start TTx DNA Polymerase

Glycerol freecomponent

carry-over prevention

Without ROX dye

carry-over prevention

Hot Start rTth DNA Polymerase
5x Buffer for rTth/ TTx (DNA/RNA)

Product nameStandard

Without ROX dye

Optional
componentStandard

Basic
component

TTx DNA Polymerase*



16

19




20
15





15





16



19

*TTx DNA Polymerase exhibits higher PCR ef iciency than Tth DNA Polymerase. The polymerase shows the reverse
transcriptase activity in the presence of Mn2+ like Tth DNA Polymerase. The polymerase enables highly sensitive andfast detection from small amount of RNA with one-step qRT-PCR and relatively tolerant of typical PCR inhibitors.

11

http://www.bio-toyobo.cn/

Raw material related Taq DNA Polymerase

rTaq DNA Polymerase Hot Start
rTaq DNA Polymerase Hot Start <Glycerol Free>
n
g soo
Comin

rTaq DNA Polymerase Hot Start is a mixture of recombinant
Taq DNA Polymerase derived from Thermus aquaticus YT-1
and anti-Taq DNA Polymerase antibodies for hot start PCR(anti-Taq high [Code No. TCP-101]). The hot start antibodies
can reduce the non-speci ic ampli ication due to preventing
mis-priming. The antibodies are inactivated at the initialdenaturation step and do not inhibit subsequent steps.
rTaq DNA Polymerase Hot Start <Glycerol Free> isglycerol-free type of rTaq DNA Polymerase Hot Start . The
reagent can be used in a preparation of master mix reagentsfor lyophilization.

Store at -20 °C
Components:

Code No. TAP-329E

10,000 U

rTaq DNA Polymerase Hot Start (5 U/l) 2 ml

Code No. TAP-359E

100,000 U

rTaq DNA Polymerase Hot Start (5 U/l) 20 ml

Code No. TAP-329GF

10,000 U

rTaq DNA Polymerase Hot Start<Glycerol Free>

Code No. TAP-359GF

2 ml

100,000 U

rTaq DNA Polymerase Hot Start<Glycerol Free>

20 ml

Features
The polymerase can be reactivated quickly compared with a chemically modified polymerase
The polymerase can be used in the reconstitution of Realtime PCR Master Mix (Code No. QPK-101).

rTaq DNA Polymerase
Taq DNA polymerase is the most widely used thermostable
DNA polymerase derived from the thermophilic bacteria
Thermus aquaticus (Taq) YT-1. The enzyme possesses a 5’→3’
polymerase activity and a double-strand speci ic 5’→ 3’
exonuclease activity.

Features
Tolerates various kinds of PCR protocols.

Store at -20 °C
Components:

Code No. TAP-201

rTaq DNA Polymerase (5 U/l)
10× PCR Buffer*
25 mM MgCl2
2 mM dNTPs

Code No. TAP-229E

Applicable for hot start technology by adding

250 U<100~200 reactions [50 l per reaction]>

10,000 U

rTaq DNA Polymerase (5 U/l)anti-Taq antibody “anti-Taq high ” (Code No.

Code No. TAP-259E

TCP-101) [p18].
PCR products can be cloned by using a TA

2 ml

100,000 U

rTaq DNA Polymerase (5 U/l)cloning method.

50 l
1 ml
1 ml
1 ml

20 ml

Code No. TAP-279E 1,000,000 U

Applicable for PCR using dUTP, dITP andrTaq DNA Polymerase (5 U/l)fluorescently-labled nucleotide.

* 100 mM Tris-HCl (pH 8.3), 500 mM KCl

200 mlApplications
PCR
Primer extension

Application data
M 1 2 3 4 5
M: 100bp Ladder
1: 180bp p53 exon8
2: 444bp p53 exon8
3: 408bp -globin
4: 1kb -globin
5: 1.3kb -globin

DW
10x PCR Buffer
2 mM dNTPs
25 mM MgCl2
10 pmol/l Primer F
10 pmol/l Primer R
rTaq DNA polymerase (5 U/l)

X (l)
55
3
1
1[1.25~2.5 U]

Template DNAY
20

94°C, 2min.
94°C, 10sec.
(Tm-5)°C, 30sec.
72°C, 1min./ kb

References
1) F.C. Lawyer, S. Stoffel, R.K. Saiki, K. Myambo, R. Drummond, D.H. Gelfand., J. Biol. Chem., 264: 6427-6437 (1989)
2) T. Nagahama, K. Sugiura, S. Lee, H. Morita, Y. Adachi, A.H. Kwon, Y. Kamiyama, S. Ikehara, Stem cells, 19: 425-435 (2001)12

http://www.bio-toyobo.cn/

25~40cyclesRaw material related Taq DNA Polymerase

Real-time PCR Reagents

n
g soo
Comin

10x Buffer for Taq Hot Start
10 x Buffer for Taq Hot Start is a concentrated buffer
components, which consists of buffer, Mg2+ and salts, used in
Realtime PCR Master Mix (QPK-101). By mixing with rTaq DNA
Polymerase Hot Start, dNTPs and ROX reference dye, QPK-101
can be reconstituted. The buffer does not contain glycerol
which inhibits lyophilization.

Store at -20 °C
Components:

Code No. QPK-1B1
10x Buffer for Taq Hot Start *20 ml

Code No. QPK-1B2
10x Buffer for Taq Hot Start *200 ml* 10x Buffer doesn’ t contain dNTPs and enzyme.

Features
The buffer can be used in the reconstitution of Realtime PCR Master Mix (Code No. QPK-101).

Raw materials can be applied to Realtime PCR. Using these raw materials related Taq DNA Polymerase, Realtime PCR
Master Mix (Code No. QPK-101) can be reconstituted. TOYOBO supplies basic and optional components so that researchers
can modify the reagents depending on the situation. Some of the components (enzymes or buffers) contain glycerol which
inhibit lyophilization. TOYOBO supply glycerol-free components.
Options

10x Buffer for Taq Hot Start













13

dNTPs













20

50x ROX reference dye











17

Uracil-DNA-Glycosylase



dUTP



12

19


20


ReverTra AceTM

Glycerol freecomponent

Reference pageOne-step RT-PCR

carry-over prevention& Glycerol freecarry-over prevention

rTaq DNA Polymerase Hot Start

Glycerol free

Without ROX dye

Optional
componentStandard

Basic
component

Product namerTaq DNA Polymerase Hot Start<Glycerol Free>

Uracil-DNA-Glycosylase<Glycerol Free>



20
12
19

13

http://www.bio-toyobo.cn/

Raw material related Tth/ TTx DNA Polymerase
Tth DNA Polymerase and TTx DNA Polymerase relatively tolerant of typical PCR inhibitor. Tth/ TTx are useful to amplify
DNA from crude samples. And these enzymes exhibit reverse transcriptase activity in the presence of Mn2+ ions. These
enzymes can be applied to one-step RT-PCR, including reverse transcription and PCR steps. These polymerase exhibits
double strand speci ic 5’ →3’ exonuclease, can be applied to TaqMan® probe assay.
Tth DNA Polymerase and TTx DNA Polymerase have higher ef iciency than rTaq DNA Polymerase. And these enzymes
relatively tolerant of typical PCR inhibitor.
Please use the following combinations for DNA ampli ication.

EnzymeSpecificity

Efficiency

Economy

Reference page

2x Buffer for Tth/ TTx(DNA) (containing
Reaction dNTP, dUTP, Mg2+)
Buffer
5x Buffer for Tth/ TTx(DNA/RNA) dNTPs
25 mM MgCl2

Hot Start rTth DNA Polymerase

+++

+++

+++

+

15

Hot Start TTx DNA Polymerase+++ ++++ +++++

15

Hot Start TTx DNA
Polymerase<Glycerol Free>

++

++

++

+++

15

Conventional Taq DNA polymerase reaction

++

++

+

lyophilization

Optional
component

Amplificaton fromcrude sample

Amplification of DNA



++

-

50x ROX reference dye

17

Uracil-DNA-Glycosylase

19

Uracil-DNA-Glycosylase <Glycerol Free>

19

dUTP

20

Tth/ TTx DNA Polymerase enable 1 enzyme one-step RT-PCR in the presence of Mn2+. Please use the following
combinations for one-step RT-PCR
TOYOBO supplies basic and optional components so that researchers can modify the reagents depending on the situation.
By using the standard option, RNA-direct™ Realtime PCR Master Mix (Code No. QRT-101, P10) can be reconstituted. Someof the components (enzymes or buffers) contain glycerol which inhibit lyophilization. TOYOBO supply glycerol-freecomponents.
Options
Tth DNA Polymerase

Reference page

carry-over prevention& Glycerol free15

5x Buffer for rTth/ TTx (DNA/RNA)













50mM Mn(OAc)2

















17

dNTPs

















20

50x ROX reference dye













17

Uracil-DNA-Glycosylase





dUTP






Hot Start TTx DNA Polymerase
Glycerol freecomponent

Glycerol freecarry-over prevention

Without ROX dye

carry-over preventionStandard

Without ROX dye

Optional
component

Hot Start rTth DNA Polymerase

Standard

Basic
component

Product nameTTx DNA Polymerase

Hot Start TTx DNA Polymerase<Glycerol free>
5x Buffer for rTth/ TTx (DNA/RNA)<Glycerol free>
Uracil-DNA-Glycosylase <Glycerol
free>

14



16

19


20
15






15





16



19

http://www.bio-toyobo.cn/

Raw material related Tth/ TTx DNA Polymerase
n
g soo
Comin

Hot Start TTx DNA Polymerase
Hot Start TTx DNA Polymerase <Glycerol Free>Hot Start TTx DNA Polymerase is a mixture of recombinant
TTx DNA Polymerase derived from Thermus sp. and anti-Taq
DNA Polymerase antibodies for hot start PCR (anti-Taq high[Code No. TCP-101]). TTx DNA Polymerase exhibits higher PCR
ef iciency than Tth DNA Polymerase, and relatively tolerant of
typical PCR inhibitors. This product can be applied toampl ication from crude samples. And this polymerase shows
the reverse transcriptase activity in the presence of Mn2+ likeTth DNA Polymerase. The polymerase enables highly sensitiveand fast detection from small amount of RNA with one-step
qRT-PCR.
The hot start antibodies can reduce the non-speci ic
ampli ication due to preventing mis-priming. The antibodies
are inactivated at the initial denaturation step and do notinhibit subsequent steps.
Hot Start TTx DNA Polymerase <Glycerol Free> is a glycerol-free type of Hot Start TTx DNA Polymerase. The reagent canbe used in a preparation of master mix reagents forlyophilization.

Store at -20 °C
Components:
2.5 mlCode No. HSTTX-129 10,000 U
Code No. HSTTX-159 100,000 U

25 ml

Code No. HSTTX-179 1,000,000 U

250 mlHot Start TTx DNA Polymerase (4 U/µl)

Code No. HSTTX-219 1,000 U

250 µlCode No. HSTTX-229 10,000 U

2.5 mlCode No. HSTTX-259 100,000 U

25 ml

Hot Start TTx DNA Polymerase (4 U/µl)<Glycerol Free>

Features
The polymerase can be reactivated quickly compared with a chemically modified polymerase
The polymerase can be used as a component of master mix reagents.
Enable one-step RT-PCR using Mn2+.
Relatively tolerant of typical PCR inhibitor.

Application data
1. Detection of enterovirus RNA
Serially (4n) diluted Enterovirus RNAs were detected using RNA-directTM Realtime PCR MasterMix based on Tth DNA Polymerase with TaqMan® probe. At the same time, the master mix
based on TTx DNA Polymerase, a mixture of Hot Start rTth DNA Polymerase, 5x Buffer for
Tth/TTx (DNA/RNA), dNTPs, ROX reference dye and Mn(OAc)2 .:[RNA-directTM Real-time PCR
Master Mix,TTx version].were tested using the same conditon. As a result, Tth version
detected 10 copies of viral RNA.
RNA-directTM Real-time PCR Master Mix (TTx version)

90℃
60℃
95℃
95℃
60℃

30sec
5min
1min
3sec
5sec

× 50

RNA-directTM Real-time PCR Master Mix

2,500 copies
2,500 copies

10 copies

Hot Start rTth DNA Polymerase
Hot Start rTth DNA Polymerase is a mixture of recombinant
Tth DNA Polymerase derived from Thermus thermophilus HB8
and hot start antibodies. The hot start antibodies can reduce
the non-speci ic ampli ication due to preventing mis-priming.
The antibodies are inactivated at the initial denaturation stepand do not inhibit subsequent steps.

n
g soo
Comin

Store at -20 °C
Components:

Code No. HSTTH-301 250 U
Hot Start rTth DNA Polymerase (4 U/µl)
2×Buffer for rTth/TTx (DNA)62.5 µl1.25 ml ×2

Code No. HSTTH-329 10,000 U

Features

Hot Start rTth DNA Polymerase (4 U/µl)

2.5 mlThe polymerase can be reactivated quickly compared with a chemically modified polymerase.
This product can be used for the reconstitution of RNA-directTM Realtime PCR Master Mix (Code No. QRT-101).

15

http://www.bio-toyobo.cn/

Raw material related Tth/ TTx DNA Polymerase

rTth DNA Polymerase

Store at -20 °C
Components:

rTth DNA polymerase is a thermostable DNA polymerase
derived from the thermophilic bacteria Thermus thermophilus(Tth) HB8.
The enzyme exhibits reverse transcriptase activity in addition
to 5’→3’ polymerase activity and double strand speci ic 5’→ 3’
exonuclease activity in the presence of Mn2+ ions: therefore,
enabling one-step RT-PCR. Kits for one-step RT-PCR (Code No.
PCR-311) and real-time PCR (Code No. QRT-101, 201, p10)
using this enzyme are available.

Code No. TTH-301

References

Code No. TTH-359L

1)T.W. Myers, D.H. Gelfand, Biochemistry, 30: 7661-7666 (1991).
2) K. Yamada, M. Terashima, M. Shimoyama, M. Tsuchiya, J Biochem. 130: 335-40 (2001)* This buffer is optimized for PCR, not one-step RT-PCR.

50 l
1 ml
1 ml
1 ml

rTth DNA Polymerase (5 U/l)
10x Buffer (contains MgCl2)*
Dilution buffer
2 mM dNTPs

Code No. TTH-329L

10,000 U
2 ml

rTth DNA Polymerase (5 U/l)

100,000 U
20 ml

rTth DNA Polymerase (5 U/l)

2x Buffer for rTth/ TTx (DNA)This product is optimized buffer for Tth/ TTx DNA Polymerase,
which contains Mg2+ and dNTP (dUTP).
This buffer provides greater ef iciency and can be applied toampli ication from crude sample. This buffer contains dUTP,
thus allowing for carryover prevention using Uracil-DNA
Glycosylase.

250 U<100~200 reactions [50 l per reaction]>

n
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Comin

Store at -20 °C
Components:

Code No. QRZ-1B1
2x Buffer for rTth/ TTx (DNA)100 mlCode No. QRZ-1B2
2x Buffer for rTth/ TTx (DNA)

Features
High specificity and effictive amplification using Tth/ TTx

Code No. QRZ-1B4
2x Buffer for rTth/ TTx (DNA)DNA Polymerase.

250 ml1000 ml* 2x Buffer contains essential components for the reaction(buffer, Mg2+, salts, dATP, dCTP, dGTP, and dUTP, etc.).

5x Buffer for rTth/ TTx (DNA/RNA)
5x Buffer for rTth/ TTx (DNA/RNA) <Glycerol Free>
n
g soo
Comin

5x Buffer for Tth/ TTx (DNA/RNA) is concentrated buffer
components, consist of buffer and salts, used in RNA-directTM
Realtime PCR Master Mix (QRT-101). By mixing with Hot Start
rTth DNA Polymerase, dNTPs, Mn2+ solution and ROX dye,
QRT-101 can be reconstituted. This buffer system can be usedwith Hot start TTx DNA Polymerase.
5x Buffer for rTth/TTx (DNA/RNA) <Glycerol Free> isconcentrated buffer components used in RNA-directTM Realtime
PCR Master Mix (QRT-101) except for glycerol which inhibit
lyophilization. This buffer can be used with Hot start TTx DNA
Polymerase <Glycerol Free> (Code No. HSTTX-229).

Store at -20 °C
Components:

Code No. QRT-1B1

40 ml

Code No. QRT-1B2

400 ml5x Buffer for rTth/ TTx* (DNA/RNA)
2 ml

Code No. QRT-2BS
Code No. QRT-2B1

20 ml

Code No. QRT-2B2

200 ml10 x Buffer for rTth/ TTx (DNA/RNA)<Glycerol Free>*

Code No. QRT-3BS

8 ml

Code No. QRT-3B1

80 ml

Code No. QRT-3B2

800 ml2.5 x Buffer for rTth/ TTx (DNA/RNA)<Glycerol Free, Contain Excipient>** Buffer doesn’ t contain Mg2+, Mn2+, dNTPs and enzyme.

Features

The buffer can be used for the reconstitution of RNA-directTM Realtime PCR Master Mix (Code No. QRT-101).
Glycerol-free buffer can be used with TTx DNA Polymerase <Glycerol Free> (Code No. HSTTX-229) for
lyophilization.

16

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Raw material related Tth/ TTx DNA Polymerase

50 mM Mn(OAc)2

Store at -20 °C

This product is used for 1 enzyme RT-PCR using Tth/ TTx DNA
Polymerase.

Components:

Code No. QRT-MN1
50 mM Mn(OAc)25 ml

Features
The reagent can be used for the reconstitution of RNA-directTM Realtime PCR Master Mix (Code No. QRT-101).

25 mM MgCl2

Store at -20 °C
Components:

This product is used for PCR.

Code No. TAP-2S1
40 ml

25 mM MgCl2

Passive reference

50x ROX reference dye

Store at -20 °C
Components:

50x ROX reference dye is used to normalize the luorescentreporter signal between the wells in real-time quantitative PCRor RT-PCR.

Code No. ROX-101
50x ROX reference dye

instruments

ROX conc.
TM

Applied Biosystems® 7000, 7300, 7700, 7900HT, StepOne , StepOnePlus

TM

1x

TM

Applied Biosystems® 7500, 7500Fast, ViiA , QuantStudio®,
0.1x

Agilent Technologies Mx3000P, Mx3005P, Mx4000

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5 mlanti-Taq DNA polymerase antibody

anti-Taq highAnti-Taq DNA Polymerase Antibody 1, 2 <Glycerol Free>
Store at -20 °C

Taq DNA polymerase

Components:
Heating step > 70℃
Polymerase activity blocked

anti -Taq antibody

Code No. TCP-101

Polymerase activity restoredanti-Taq high is a highly puri ied neutralizing monoclonal
antibody to Taq and Tth DNA polymerase. This productprovides an antibody-mediated hot start PCR to enhance thespeci icity and sensitivity of PCR. This antibody inhibits
polymerase activity before the onset of thermal cycling,
preventing primer dimer formation and non-speci ic
ampli ication. At the irst denaturation step of the thermal
cycling, the antibodies are quickly inactivated and PCR
proceeds. The antibody-mediated hot start method issigni icantly more convenient to use than other hot start
methods.
anti-Taq high contains two antibodies. Anti-Taq
DNA Polymerase Antibody 1, 2<Glycerol Free> are glycerol-free
antibodies of anti-Taq high.

anti-Taq high (1 mg/ml)*
10x PCR buffer

100 l1 ml

Code No. TCP-139
anti-Taq high (1 mg/ml)*

30 ml

Code No. TCP-189CH1
anti-Taq high (1 mg/ml)*

100 mlCode No. TCP-309
Anti-Taq DNA Polymerase Antibody 1<Glycerol Free>**

1 mg

Code No. TCP-319
Anti-Taq DNA Polymerase Antibody 1<Glycerol Free>**

25 mg

Code No. TCP-409

FeaturesAnti-Taq DNA Polymerase Antibody 2<Glycerol Free>**Enhances the specificity and sensitivity of PCR.
Inhibits ≥95% of Taq DNA polymerase activity with

1 mg

Code No. TCP-419

anti-Taq high at 45ºC.

Anti-Taq DNA Polymerase Antibody 2

No contamination of mouse genomic DNA, as<Glycerol Free>**

25 mgdetermined by PCR.
The polymerase can be reactivated quickly comparedwith a chemically modified polymerase.

* Storage buffer: 10 mM Tris-HCl (pH 7.0), 50 mM KCl, 50% Glycerol** Storage buffer: 20 mM Tris-HCl (pH 7.5)
The concentration is different depending on Lot No.

Application data

1 2 3 M

1. Application of the hot start PCR using a Taq-based highefficient PCR enzyme
The efficiency of anti-Taq antibodies were evaluated by the
amplification of the human β-globin gene (3.6 kb). The result
indicates that anti-Taq high increases the specificity andsensitivity of the PCR compared with the control reaction
and PCR mediated hot start using company A's anti-Taq
antibody.

M: λ/Hind III Marker
1: Taq-based high efficient PCR enzyme
2: Taq-based high efficient PCR enzyme + anti-Taq high
3: Taq-based high efficient PCR enzyme + anti-Taq
antibody (company A)

anti-Taq Neo
anti-Taq Neo <Glycerol Free>anti-Taq Neo and anti-Taq Neo<Glycerol Free> are basic
version of anti-Taq high. This products provide an
antibody-mediated hot start PCR to enhance the speci icity
and sensitivity of PCR to neutralize Taq DNA Polymerase.
anti-Taq Neo <Glycerol Free> is glycerol-free type of anti-Taq
Neo.

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Store at -20 °C
Components:

Code No. TCP-239
anti-Taq Neo (1 mg/ml)

30 ml

No contamination of mouse genomic DNA, as

Code No. TCP-259
anti-Taq Neo (1 mg/ml)

100 mldetermined by PCR.

Code No. TCP-219GF

Enhances the specificity and sensitivity of PCR.
Inhibits ≥90% of Taq DNA polymerase activity at 45ºC.

The polymerase can be reactivated quickly comparedanti-Taq Neo <Glycerol Free>

with a chemically modified polymerase.

18

http://www.bio-toyobo.cn/

25 mg

Recombinant type RNase inhibitor

RNase Inhibitor, RecombinantThis product is a recombinant human placental ribonucleaseinhibitor.
This inhibitor exhibits broad-spectrum RNase inhibitory
properties, including RNase A, RNase B, RNase C and humanplacental RNase, does not inhibit RNase T1, S1 nuclease,
RNase from Aspergillus and RNase H. This inhibitor (51 kDa)
exerts its inhibitory effect by noncovalently binding to RNases
in a 1:1 ratio. This inhibitor can be applied to a reverse
transcriptase reaction.

Store at -20 °C
Components:

Code No. SIN-201 2,500 U
Code No. SIN-229 100,000 U
Code No. SIN-259 1,000,000 U
RNase inhibitor (20-40 U/l)
One unit is defined as the amount of RNase inhibitor
required to inhibit the activity of 5 ng of ribonuclease A
by 50%.

Applications
cDNA synthesis, RT-PCR

Application data
1. Comparison of inhibitory effect between different types of RNase inhibitors

Cell lysates were prepared from 2.5 x 103 U937 cells in thepresence of human placenta (TOYOBO), porcine and murine
recombinant RNase inhibitors. Then, cDNA weresynthesized using the lysates and -actin genes wereanalyzed by real-time PCR.
The cDNA synthesized from the lysate with human placental
RNase inhibitor showed better results than that with other
RNase inhibitors.

Human placental RNase inhibitor (TOYOBO)
Porcine RNase inhibitor
Murine RNase inhibitor
No inhibitor (not detected)

Uracil-DNA Glycosylase

Uracil-DNA Glycosylase, Heat-labile
Uracil-DNA Glycosylase, Heat-labile<Glycerol Free>

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Store at -20 °C
Components:

Uracil-DNA Glycosylase, Heat-labile can be used with dUTP toeliminate carryover contamination from previous DNA
synthesis reactions.
This enzyme is completely and irreversibly inactivated bymoderate heat treatment (55°C) and does not degrade PCR
products after PCR.
Uracil-DNA Glycosylase, Heat-labile <Glycerol Free> isglycerol-free type of Uracil-DNA Glycosylase, Heat-labile. The
reagent can be used in a preparation of master mix reagentsfor lyophilization.

Code No. UNG-101 200 U
Code No. UNG-109 10,000 U
Uracil-DNA Glycosylase, Heat-labile (1 U/l)

Code No. UNG-201 200 U
Code No. UNG-209 10,000 U
Uracil-DNA Glycosylase, Heat-labile<Glycerol Free>

Applications
One unit of UNG is defined as the amount of enzyme
required to release 1 nmol uracil from uracil-containing
DNA per hour at 37°C.

Carryover prevention

19

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High Efficient Reverse Transcriptase

ReverTra AceTM

Store at -20 °C
Components:

ReverTra Ace is a high ef icient M-MLV (Moloney Murine
Leukemia Virus) reverse transcriptase that has been
genetically modi ied to reduce RNase H activity and increase
reaction ef iciency. It is the preferred enzyme for applications
requiring full-length cDNAs and high product yields from total
RNA, mRNA, rRNA, etc.
TM

Code No. TRT-101
ReverTra Ace
5x BufferTM

10,000 U
100 l1 ml(100 U/l)

Code No. TRT-109

500,000 U

ReverTra AceTM (100 U/l)

5 ml

Features
RNase H minus M-MLV RTase with improved performance.
Enables the synthesis of longer cDNAs (≥ 14 kb).

One unit is defined as the amount of enzyme
required to incorporate 1 nmole of dTTP into an
acid-insoluble material in 10 min at 42ºC.

Excellent reaction efficiency at high temperatures.
Higher reverse transcription efficiency Decrease RNase H activity-

M-MLV RTase

ReactionTM
Company A
temperature ReverTra Ace(°C)
42 50 55 60 42 50 55 60

Polymerase domain

RNase H domain

RTase activity

RNase H activity

9.49
7.46
4.40
2.37

Prevent ion of degradingof the mRNA

1.35

Enhancement of the cDNA
synthesis ability
AAAAAAAAAA
TTTTTTTTTT

Company B
42 50 55

Fig. Comparison of elongation capability of RNase H
minus RTases at various temperatures

Fig. Genetically improved M-MLV Reverse transcriptase

Nucleotides

dNTP (dATP, dCTP, dGTP,dTTP, dUTP)
dNTPs MixturedNTPs Set
Store at -20 °C
Components:

Code No. NTP-101
dATP, dCTP, dGTP, dTTP (100 mM), 0.5 ml eachdNTPs Mixture is an equal mole solution mixture ofultrapure dATP, dCTP, dGTP, and dTTP, or dATP, dCTP,
dGTP, and dUTP. dNTPs Set contains each dATP, dCTP, dGTP,
and dTTP solution.

Code No. NTP-201
dNTPs Mixture (A, C, G, T each 2 mM), 1 ml

Code No. NTP-301
dNTPs Mixture (A, C, G, T each 10 mM), 0.2 ml

Applications

Code No. NTP-501
PCR

dNTPs Mixture (A, C, G, U each 2 mM), 1ml

Reverse transcription

Code No. ATP-109
dATP (100 mM), 100 ml

Code No. CTP-109
dCTP (100 mM), 100 ml

Code No. GTP-109
dGTP (100 mM), 100 ml

Code No. TTP-109
dTTP (100 mM), 100 ml

Code No. UTP-101
dUTP (100mM), 500μl

20

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Efficient heat-stable T7 RNA polymerase

Thermo T7 RNA Polymerase
Thermo T7 RNA Polymerase is a genetically modi ied T7 RNApolymerase exhibiting increased thermal stability. The
optimum reaction temperature of this enzyme is around 50 ºC.
The half-life of the enzyme at 50 ºC is approximately 85 min.

Store at -20 °C
Components:

Code No. TRL-201

7,500 U

Thermo T7 RNA Polymerase (50 U/l)
10x Buffer*

Code No. TRL-252

150 l2 x 1ml50,000 U

Thermo T7 RNA Polymerase (1,000 U/l) 50 l
3 x 1ml
10x Buffer** The following reagents are not supplied;
NTP and RNase inhibitor.

Features

Increased enzyme activity compared with wild type enzyme at 37-50ºC.

Applications
RNA probe preparation
RNA synthesis for in vitro translation
RNA synthesis for RNA splicing studies
Capped mRNA synthesis using a cap analogue

Unit definition
One unit is defined as the amount of enzyme that will incorporate 1 nmole of [3H] rNTP into an acid insoluble materialusing T7 DNA as template in 60 min at 37ºC.

% Retained activity

Application data
1. Comparison of heat stability
The residual activities of the wild-type T7 RNA Polymerase andThermo T7 RNA polymerase were measured after incubation forvarious periods at 48ºC and 50ºC, respectively. As a result,
Thermo T7 RNA polymerase retained its activity after incubation for
15 min whereas the activity of wild-type enzyme decreased to 1/10
after incubation for 5 min.

100
10
1
0

5

10

15

20

Incubation time (min)The half-lives of the enzymes have been estimated to be:
Wild-type T7 RNA polymerase: < 1.9 min. (at 48ºC.)
Thermo T7 RNA Polymerase: 84.5 min. (at 50ºC.)

2. In vitro transcription at high temperature

Wild-type T7 RNA polymerase (at 48ºC)
Thermo T7 RNA polymerase ( at 50ºC)

Reaction
temperature (ºC)

In vitro transcriptions were performed using wild
type and Thermo T7 RNA Polymerases at various
temperature conditions (37, 45, 48, 51ºC.).
Template plasmid
The transcripts were detected by agarose gel
Transcript
electrophoresis. Distinct transcripts were detectedfrom 37 to 51ºC with Thermo T7 RNA Polymerase.

Wild-type
T7 RNA pol.

ThermoT7 RNA pol.

37 45 48 51 M 37 45 48 51

Template:
Linearlized plasmid DNA carrying T7 promoter, 0.5 mg
Reaction time: 60 min.

21

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High success-rate PCR enzyme

KOD OneTM PCR Master Mix
KOD OneTM PCR Master Mix -BlueStore at -20 °C
Components:

Code No. KMM-101<200 reactions [50 μl per reaction]>
KOD OneTM PCR master Mix and KOD OneTM PCR Master Mix-Blue- are 2 x PCR master mixes based on geneticallymodi ied KOD DNA polymerase (UKOD). KOD OneTM series
enables fast PCR, which has an extension time of 5 sec/ kb
by applying UKOD and a new Elongation Accelerator. In
addition, these master mixes provide greater ef iciency andelongation capabilities than conventional PCR enzymes. In
particular, they show greater ampli ication success fromcrude specimens. Furthermore, these master mixes can beapplied to amplify from templates containing uracils (dU) or
using primers containing inosines (dI) and uracils (dU).

KOD OneTM PCR Master Mix

1 ml x 5

Code No. KMM-201<200 reactions [50 μl per reaction]>
KOD OneTM PCR Master Mix -Blue- 1 ml x 5*The reagents can be stored at 4°C for a month. For longer
storage, the reagents should be kept at -20°C

KOD OneTM series contains two types of anti-KOD DNA
polymerase antibodies that inhibit the polymerase and 3’→
5’ exonuclease activities, thus allowing for Hot Start PCR.
These master mixes generate blunt-end PCR products
because of 3’ → 5’ exonuclease (proof-reading) activity of
KOD DNA polymerase.

Featuresｰ Fast
KOD OneTM series can amplify the targets using the following very short conditions:≤1 kb: 1 sec
1~ 10 kb: 5 sec/ kb
10 kb~: 10 sec/ kb
The cycling conditions can be set flexibly when various targets having different sizes are amplified.
- Easy to Use
KOD OneTM series contains all reaction components except for primers and templates and provide highreproducibility by reducing operations. In addition, KOD OneTM PCR Master Mix -Blue- includes
a loading dye (BPB) to allow direct loading onto agarose gels.
- High Fidelity
KOD OneTM series exhibits approximately 80-fold higher fidelity than Taq DNA polymerase. These
mixes can be used for various purposes where this would be an advantage (e.g., in the preparationof long target amplicons for sequencing).
- High Efficiency
KOD OneTM series is effective for amplification from crude samples (e.g., biological samples, foodstuffs,
soil extract, etc.). Various samples or lysates can be used directly as templates.

Applications
Direct PCR
Colony PCR
Amplification of NGS libraries
Site-directed gene mutation

22

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High success-rate PCR enzyme

Application data
1. Fast PCR
Various targets were amplified with
KOD OneTM PCR Master Mix and
KOD OneTM PCR Master Mix -Blueusing the fast cycling conditions.
KOD OneTM series successfully
amplified all targets.

<Reaction Mix>
distilled water
KOD OneTM PCR Master Mix
10pmol/µl Primer F
10pmol/µl Primer R
10ng/µl human genomic DNA

21µl
25µl
1.5µl
1.5µl
1μl

Total Volume

50µl<PCR cycle>
Target length <1kb
98℃
10sec
60℃
5sec
X30 cycles
1sec
68℃

Target length 1-10kb
98℃
10sec
60℃
5sec
X30 cycles
68℃
5sec/kb

Template: human genomic DNA[Target length <1kb: Extension 1 sec 1-10 kb: Extension 5 sec/kb]
KOD OneTM
PCR Master Mix

KOD OneTM
PCR Master Mix

M 1 2 3 4 5 6 7 8 9 10

M ： 100bp DNA Ladder
1 ： DDB2(200bp)
2 ： FANCG(250bp)
3 ： HBg(300bp)
4 ： CDH1(400bp)
5 ： chrome9(500bp)

6 ： ERCC4(550bp)
7 ： HRAS(600bp)
8 ： PRF1(700bp)
9 ： BRCA1(800bp)
10 ： CDK4(1,000bp)

Company A
PCR Master Mix

M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7

M ： 1kb DNA Ladder
1 ： Chrome9(1kb)
2 ： MSH6(2kb)
3 ： FANCE(3kb)

4 ： RAD51D(4kb)
5 ： KRAS(5kb)
6 ： BRCA1(7kb)
7 ： DDB2(10kb)

2. Amplification efficiency and sensitivity
The 3.5 kb fragments were amplified from plasmid DNA andcDNA. Each PCR reaction was performed according to therecommended conditions. KOD OneTM PCR Master Mix
showed higher sensitivity using 5 sec/kb extension time.

[plasmid DNA］

<Reaction Mix>
distilled water
KOD OneTM PCR Master Mix
10pmol/µl Primer F
10pmol/µl Primer R
template

21µl
25µl
1.5µl
1.5µl
1μl

Total Volume

50µl<PCR cycle>
98℃
10sec
60℃
5sec
68℃
18sec (5sec/kb)［cDNA］

Template: plasmid DNA
Target: Polymerase gene

KOD OneTM
PCR Master Mix

Template: cDNA from HeLa total RNA
Target: TFR

Company A
PCR Master Mix

KOD OneTM
PCR Master Mix

M 1 2 3 4 M 1 2 3 4Company A
PCR Master Mix

M 1 2 3 4 M 1 2 3X30 cycles

3.5kb
3.5kb
4

M
1
2
3
4

： 1kb DNA Ladder：25 pg
6.3 pg：：
1.6 pg：0.4 pgM
1
2
3
4： 1kb DNA Ladder： 25 ng equivalent： 5 ng equivalent： 1 ng equivalent： 0.2 ng equivalent

3. Amplification using degenerate primers containing inosine.
The 2.8 kb fragments were
amplified using degenerate primers
containing inosine. KOD OneTM PCR
Master Mix was able to amplify,
2.8kb
whereas conventional high-fidelity
PCR enzymes were not.

M 1 2 3
＜Reaction Mix＞
distilled water
KOD OneTM PCR Master Mix
100pmol/µl Primer F
100pmol/µl Primer R
50ng/µl E.coli genomic DNA

17µl
25µl
1.5µl *
1.5µl *
5μl

Total Volume

50µl＜PCR cycle>
98℃
10sec
5sec
60℃
68℃
15sec

X30 cycles<Primer sequence>
Fwd: ATGGTICARATHCCICARAAY
R e v : RTGIGCYTGRTCCCARTTYTC*In cases where degenerate primers are used, the primer concentrations should be increased
depending on the degree of degeneracy.
M ： 1kb DNA Ladder
1 ： KOD OneTM PCR Master Mix

23

2
3

： KOD OneTM PCR Master Mix -Blue： KOD -Plus- Neo

http://www.bio-toyobo.cn/

High success-rate PCR enzyme

Application data
4. Amplification from cDNA
KOD OneTM
PCR Master Mix

The inhibitory effect of RNA in cDNA wascompared using various PCR enzymes.
KOD OneTM PCR Master Mix was not
susceptible to RNA inhibition, and it wasable to amplify targets under highconcentrations of cDNA.

M

2

3

4

M

1

2

3

KOD -PlusNeo

4

M

1

2

3

KOD FX
Neo

4

M

1

2

3

4

3.5kb

<Reaction Mix>
distilled water
KOD OneTM PCR Master Mix
10pmol/µl Primer F
10pmol/µl Primer R
cDNA

17µl
25µl
1.5µl
1.5µl
5μl

Total Volume

50µl＜PCR cycle＞
98℃
10sec
60℃
5sec
18sec (5sec/kb)
68℃

1

KOD OneTM
PCR Master Mix -Blue-

Company A
PCR Master Mix

M

1

2

3

4

Company A
PCR Kit

M

1

2

3

Company B
PCR Kit

4

M

1

2

3

Company C
PCR Kit

4

M

1

2

3

4

3.5kb

X30 cycles

The PCR was performed according to
the manufacturerʼs recommendations.
M ： 1kb DNA Ladder3 ： 500 ng(RNA equivalent)
1 ： 125 ng(RNA equivalent) 4 ： 1,000 ng(RNA equivalent)
2 ： 250 ng(RNA equivalent)

Template: cDNA from HeLa
total RNA
Target:TFR

5. Amplification efficiency and sensitivity［Ampliﬁcation from mouse lysate］

Amplification from mouse lysate and whole blood werecompared. KOD OneTM PCR Master Mix amplified thetargets efficiently.
1

KOD OneTM
PCR Master Mix

2

M 1

mouse tail（approx. 3mm）Transfer tomicrotube

2

3

4Company A
PCR Master Mix

M 1

2

3

4

2.6kb

3
50mM NaOH 180l▼Template: supernatant
M：1kb DNA Ladder
1：1 µl 2：2 µl 3：4 µlvortex well4

4：8 µl［Direct ampliﬁcation from

95℃ for 10min▼

whole blood］

1M Tris-HCI(pH8.0) 20l▼

KOD OneTM
PCR Master Mix

vortex well▼

12,000rpm, 5min.[optional]

M 1

5

2

3

4

Company A
PCR Master Mix

1

2

3

3.6kb

0.5-2l of supernatant▼

PCR(50l)＜Reaction Mix＞
distilled water
KOD OneTM PCR Master Mix
10pmol/µl Primer F
10pmol/µl Primer R
template

Ｙµl
25µl
1.5µl
1.5µl
Xμl

Total Volume

50µl＜PCR cycle＞
98℃
10sec
60℃
5sec
68℃
5sec /kb

X30 cycles

Template: whole blood
M：1kb DNA Ladder
1：1 µl 2：2 µl 3：4 µl 4：8 µl

The PCR was performed according to the manufacturerʼs recommendations.

24

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High success-rate PCR enzyme

KOD FXStore at -20 °C
Components:

Code No. KFX-101 200 U<200 reactions [50 μl per reaction]>
KOD FX is based on DNA polymerase from thehyperthermophilic Archaeon Thermococcus kodakaraensis
KOD1(1)(2). KOD FX results in much greater PCR successbased on ef iciency and elongation capabilities than KOD-Plus- or other Taq-based PCR enzymes. KOD FX enzyme
solution contains two types of anti-KOD DNA polymerase
antibodies that inhibit polymerase and 3’5’ exonucleaseactivities, thus allowing for Hot Start PCR(3). KOD FX
generates blunt-end PCR products, due to 3’5’exonuclease (proof-reading) activity.

KOD FX (1.0 U/l)*
2 x PCR Buffer for KOD FX
2 mM dNTPs

200 l3 x 1.7 ml
2 x 1 ml

*The enzyme solution contains anti-KOD DNA
polymerase antibodies that neutralize polymerase and3’5’ exonuclease activity for hot start technology.

Features
Effective amplification from crude samples.
Direct amplification from various samples (whole blood, microorganisms).
Effective amplification of difficult targets, such as high GC or AT content, and/or long targets.
Effective amplification of long target.
The following amplifications is confirmed:
40kb from phage lambda DNA, 24kb from human genomic DNA, and 13.5kb from cDNA.
Low error ratio, about 10 times less than that of Taq DNA polymerase.
PCR products amplified using KOD FX is of blunt-end. TArget Clone™ -Plus- can be applied to the TA cloning.

Applications
Genotyping (Amplification from crude samples)
Efficient amplification of difficult targets (high G/C, long ) from genomic DNA or cDNA with fast mode
High fidelity PCR

Application data
1. Comparison of PCR efficiency on mouse genotyping
The target loci of the transgenic mice were amplified from the mouse tail lysates [see p13 Alkaline lysis methods] using
KOD FX and the other Taq-based high efficient PCR enzymes. The target loci (3.1 kb) were successfully amplified by KOD
FX.
Taq based high efficient
Sample: Supernatant of mouse tail lysates 1 l / 50 l Reaction
KOD FX
PCR enzyme
Targeted locus (Tg)loxP

PGK/puro loxP

Primer F

1 2 3 M 1 2 3 M 1 2 3Primer R

3.1kb
1.5kb

[Cycling conditions of KOD FX ]94°C, 2min.
98°C, 10sec.
68°C, 2 min.

M: 1kb DNA Ladder
1: Tg / Tg
2: Tg / W
3: W / W

30 cycles

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)25

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Protocols for amplification from crude samples using KOD FX
KOD FX is suitable for ampli ication from crude specimens such as whole blood, various lysates (e.g. mouse tail, plant
samples) and organisms bearing cell walls (e.g. yeast, fungi, gram-positive bacteria). We recommend the following
protocols for ef icient ampli ication.

Direct amplification from whole blood

Amplification from mouse tail lysates
Alkaline lysis method

Anticoagulant
Citric acid
HeparinM 1 2 3 4M 1 2 3 4Transfertomicrotube

Mouse tail（Approximately
3mm）

Direct1
1: KOD FX
2: KOD FX Neo
3: PCR enzyme (A company)
4: PCR enzyme (B company)
M: 1kb DNA Ladder

Whole blood
with an anticoagulant
・EDTA
・Citric acid・Heparin

PCR

2

50 mM NaOH 180 l

3DW
X (l)
2x PCR Buffer
25
2 mM dNTPs
1010 pmol/ml Primer F
1.5
10 pmol/ml Primer R
1.5
KOD FX or KOD FX Neo (1U/ml) 1
Whole blood / Lysates etc.
1~2*

95˚C for 10 min.

Total

Supernatant 0.5-2 l

1 M Tris-HCl (pH 8.0) 20 l

4Vortex well
12,000 rpm, 5 min. [optional]

50*In the case of microorganisms colony, the sample
volume should be omitted.

5

PCR (50 l)

Amplification from plant samples
Homogenization method

One step method

This protocol is effective for the amplification of the targets
on genomic DNA

2

1
Leaf
Rice grain（3x3mm）

2

1

Transfertomicrotube

Transfertomicrotube

Leaf（3x3mm）

3

Buffer A 100 μl
Vortex

Buffer A 100 μl

Buffer A*:
100mM Tris-HCl(pH9.5)
1M KCl10mM EDTA

Vortex4

95℃ for 10 min.

3

Buffer A*:
100mM Tris-HCl(pH9.5)
1M KCl10mM EDTA

4Vortex well

Homogenize using pestle
Supernatant 1 μl

5PCR (50 μl)

Supernatant 1 μl

5PCR (50 μl)

Left: Rice leaf
Right: Rice grain*BioTechniques, 19: 394 (1995)*BioTechniques, 19: 394 (1995)

Direct amplification from yeast & fungus colonies
PCR

KOD FX

Company A

M 1 2 3 4 5

M 1 2 3 4 5

M: 100 bp DNA Ladder
1 Aspergillus oryzae
2 Aspergillus niger
3 Saccharomyces cerevisiae
5 Schizosaccharomyces pombe
6 Pichia pastris

Direct26

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NGS related product

GenNextTM NGS Library Prep Kit
Store at -20 °C
Components:

Code No. LPK-101T 8 reactions
LPK-101 24 reactions
LPK-101L 96 reactions
End Repair and A-tailing Buffer
End Repair and A-tailing Enzyme
Ligation Solution
Library Amplification Master Mix
Library Amplification Primer Mix
50x Dilution Buffer

The GenNext NGS Library Prep Kit designed for the rapid
construction of libraries for Illumina® sequencing, fromfragmented double-stranded DNA(dsDNA).
The GenNextTM NGS Library Prep Kit is designed for Library
construction from a wide range of sample inputs (1ng-1μg).
TM*Adapter and SPRI (Solid Phase Reversible Immobilization)
paramagnetic bead are not included in this kit.

The kit contains all of the enzymes and reaction buffers
required for end repair and A-tailing, adapter ligation, andlibrary ampli ication(optional).

Features
The kit performs end repair and A-tailing with an enzyme mix in a single tube.
The kits are designed for library construction from a wide range of sample types and inputs (1 ng – 1 μg).
Library Amplification Master Mix is designed for low-bias, high-fidelity PCR.

Applications
Library Preparation of Illumina® next-generation sequences

Application data
1. Comparison of NGS accuracy with other manufacturers kit

93

Nucleotide differences

Mapped reads (%)

95

91
89
87
1μg

1ng

in reads relative to reference (%)

We performed sequencing of E. coli genomic DNA using MiSeq and MiSeq Reagent Kit v2 (300 Cycles). CLC
Genomics Workbench (Qiagen/CLC bio) was used for the sequence analysis, after the downsampling of eachsamples for 1 million leads.
GenNextTM Library Prep Kit provides superior results of mapped reads and matching rate with the reference.

95
94.5
94
93.5
93
1μg

PCR 12cycles

1ng
PCR 12cycles

GenNextTM NGS Library Prep Kit

27

Company A

http://www.bio-toyobo.cn/

NGS related product

GenNextTM NGS Library Quantification Kit
Store at -20 °C
Components:

Code No. NLQ-101

GenNextTM NGS Library Quanti ication Kit is for the SYBR®
Green I qPCR-based library quanti ication of Illumina
next-generation sequences. The kit allows the speci ic andaccurate quanti ication of libraries bearing P5 and P7
adaptors which can be applied to low cell ampli ication. It
uses the highly ef icient qPCR master mix KOD SYBR® qPCR
Mix.

500 reactions

KOD SYBR® qPCR Mix
50x ROX reference dyeStandard DNA 1 (20 pM)
Standard DNA 2 (2 pM)
Standard DNA 3 (0.2 pM)
Standard DNA 4 (0.02 pM)
Standard DNA 5 (0.002 pM)
Standard DNA 6 (0.0002 pM)
5x Primer Mix
50x Dilution Buffer

3 ｘ 1.67 ml
250 l200 l200 l200 l200 l200 l200 l2 x 1 ml
1.7 mlFeatures
KOD SYBR® qPCR Mix can efficiently amplify GC- and AT-rich fragments of different lengths without bias.
The kit has a broad dynamic range from 20 pM (Standard DNA 1) to 0.0002 pM (Standard DNA 6).
The kit contains all reagents (KOD SYBR® qPCR Mix, 5X Primer Mix, Standard DNA, and 50X Dilution buffer)needed for the qPCR-based quantification of an NGS library.

Applications
qPCR-based library quantification of Illumina next-generation sequences

Application data
1. Comparison of PCR amplification efficiency of target genes with various GC contents.
Template DNAs of various GC contents bearing P5 andP7 adaptor sequences were analyzed. The GenNextTM
NGS Library Quantification Kit achieved stable PCR
efficiency with all targets, whereas other products
showed poor specificity for targets with high GC
contents.

PCR Efficiency (%)

100

90

80

70

*
Standard DNA**of each company

* *

27.3%

69.5%

80.3%

83.0% [GC content](296 bp)(450 bp)(294 bp)(188 bp)

GenNextTM NGS Library Quantification Kit(Size)*Not determined

Company A
Company B** Our standard DNA: 452 bp, GC content: 53.1%

2. Comparison of PCR amplification efficiency of target genes with various fragment sizes

PCR Efficiency (%)

100

Template DNAs of various fragment sizes (150–800bp) bearing P5 and P7 adaptor sequences wereanalyzed. The GenNextTM NGS Library Quantification
Kit achieved stable PCR efficiency with all targets,whereas other products showed poor efficiency withlong targets.

90

80
Standard DNA* 150 bp
of each company (56.6%)

300 bp400 bp600 bp800 bp[ Size ](46.5%)(34.4%)(39.3%)(39.3%)(GC content )GenNextTM NGS Library Quantification Kit
Company A
Company B*Our standard DNA: 452 bp, GC content: 53.1%

28

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订货相关咨询
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2019.3.1