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Instruction manual for Hot Start TTx (DNA) Kit 2007

F1787KHot Start TTx (DNA) Kit
HSTTX-101

250U

Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Protocol
1. Standard reaction setup
2. Cycling conditions[4]

Example[5]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory
precautions and safety while using this kit.
-TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
-This product is sold in U.S. under the license of US patent 7772383 from Chakrabarti Advanced Technology NewCo LLC.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(86)-21-5879-4900

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

1

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

Description
Hot Start TTx (DNA) Kit is PCR reagent based on our original polymerase, TTx DNA
Polymerase. TTx DNA Polymerase has higher amplification efficiency than Taq DNA
Polymerase, which is a general-purpose enzyme, and enables amplification using fastcycle condition and amplification from a crude sample containing PCR inhibitors.
In addition, TTx DNA Polymerase has a 5 '- 3' exonuclease activity, so it can be used forreal-time PCR using probe assays such as TaqMan® assay. This enzyme contains
neutralizing antibodies, thus allowing for Hot start PCR.

Features-Excellent DNA Amplification Efficiency
The reaction composition is optimized based on TTx DNA Polymerase. TTx DNAPolymerase has higher elongation capacity than general-purpose enzymes such as Taq
DNA Polymerase and Tth DNA Polymerase, TTx DNA Polymerase enable efficient PCReven fast cycle condition.
- Tolerant of PCR Inhibitors
This kit is effective for amplification from crude samples (e.g., biological samples,
foodstuffs, soil extract, etc.). In the case of amplification from whole blood, sufficient
amplification can be achieved by adding it directly to the reaction solution without
purification of nucleic acid.
- Utilization of dUTP
This kit contains dUTP instead of dTTP in 2x Buffer for rTth/ TTx (DNA). Therefore,
the rate of false-positive detection can be reduced by adding uracil-N-glycosylase(UNG).
*UNG is not supplied with this kit.

[2] Components
This kit includes the following components for 250 reactions, 20 μL total reaction
volume. All reagents should be stored at -20°C.
2x Buffer for rTth/ TTx (DNA)Hot Start TTx DNA Polymerase (4U/ μL)

1.25 mL x 2
62.5 μL

Note:-2x Reaction Buffer contains essential components for the reaction (buffer, salts, Mg2+,dATP, dCTP, dGTP, and dUTP, etc.). Add template DNA, primers, and attached Hot Start
TTx DNA Polymerase, and adjust to 1x concentration with sterile water etc.
-DNA Polymerase is a mixture of TTx DNA polymerase and hot start antibodies. Its
concentration is 4U/ μL.
-This kit doesn’t contain a passive reference dye (ROX). When using a passive reference
dye to compensate fluorescence intensity and dispensing error between wells, please use
the separately sold 50x ROX reference dye (Code No. ROX-101).

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

CHINA
TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(86)-21-5879-4900

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

2

www.toyobo.co.jp/e/bio[3] Protocol

1. Standard reaction setup
Before preparing the mixture, all components should be completely thawed, except for
the enzyme solution.
Components
PCR grade water
2x Buffer for rTth/ TTx (DNA)
10 µM Primer #1
10 µΜ Primer #2
TaqMan® Probe(10 μM)
Hot Start TTx DNA Polymerase
Template DNA (Sample)
Total reaction volumeVolumeX µL
10 µL
0.6 µL0.6 µL0.4 µL0.25 µLY µL
20 µL

Final Concentration
1x0.3 µM
0.3 µM
0.2 µM
1UNotes:-The recommended amount of primer should be 0.2-0.6 μM, and the amount ofTaqMan® probe should be 0.05-0.3 μM. If amplification efficiency is not good,
performance may be improved by increasing the addition amount, but if it is added toomuch, it may cause non-specific reaction and detection sensitivity may be lowered.

2. Cycling conditions
The following cycle is recommended.
Temperature

TimePredenature :95°C

1 min.

Denature :95°C

15 sec.

Annealing/extension :60°C

30 sec.

40~50 cycles

Notes:[Optional] The uracil-N-glycosylase (UNG) treatment step should be added before
predenature step. The indicated temperature and time are typical conditions for UNG.
The conditions can be optimized according to the particular instruction manual fromthe supplier of UNG.
- If sensitivity is not good, it may be improved by changing annealing/ extension
temperature between 55 ~ 65°C.

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

CHINA
TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(86)-21-5879-4900

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

3

www.toyobo.co.jp/e/bio[4] Example

Detection of african swine fever virus
African swine fever virus DNA was detected using TaqMan® probes. As a result ofperforming in 20 μL reaction mixture with and without 2.5 μL of plasm, Taq DNA
Polymerase-based reagent was inhibited by plasma and the amplification could not be
confirmed. On the other hand, Hot Start TTx (DNA) Kit was able to detect african swine
fever virus DNA even adding plasma.

[5] Related products
Product namePackage

Code No.

<High efficient DNA Polymerase for PCR and RT-PCR>

10,000 U

HSTTX-129

Hot Start TTx DNA Polymerase

100,000 U

HSTTX-159

100 mL

QRZ-1B1

250 mL

QRZ-1B2

1,000 mL

QRZ-1B4

5 mL

ROX-101

10,000 U

HSTTH-329< Reaction Buffer (not containg Mg2+ and Mg2+>

40 mL

QRT-1B1

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

400 mLQRT-1B25mL

QRT-MN1

40 mL

TAP-2S1<Reaction Buffer (containg Mg2+) for DNA amplification>

2x Buffer for rTth/ TTx (DNA)<Passive reference dye>

50x ROX reference dye<High efficient DNA Polymerase for PCR and RT-PCR>

Hot Start rTth DNA Polymerase<Mn solution for RNA amplification>

50 mM Mn (OAc)2<Mg solution for DNA amplification>

25 mM MgCl2

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

CHINA
TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(86)-21-5879-4900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
4