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Instruction manual for Hot Start rTth (DNA) Kit 2103

F1768KHot Start rTth (DNA) Kit
HSTTH-301

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.

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 rTth (DNA) Kit is PCR reagent based on Tth DNA Polymerase. Tth DNA
Polymerase has higher amplification efficiency than Taq DNA Polymerase, which is a
general-purpose enzyme, and enables amplification from low copies of template andamplification from a crude sample containing PCR inhibitors.
In addition, Tth 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 Tth DNA Polymerase, enabling
efficient PCR even from low copies of template.
- 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 rTth DNA Polymerase (4U/ μL)

1.25 mLx 2
62.5 μL

Note:-2× 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
rTth DNA Polymerase, and adjust to 1x concentration with sterile water etc.
-DNA Polymerase is a mixture of rTth 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 rTth DNA Polymerase
Template DNA (Sample)
Total reaction volume

Volume
X µL
10 µL
0.6 µL0.6 µL0.4 µL0.25 µL
Y 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

Time

Predenature :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 SNPs
SNPs in aldehyde dehydrogenase 2 (ALDH2) gene were detected using TaqMan® probes.
1 μLof whole blood was added to 20 μLreaction mixture as template. As a result, Taq
DNA Polymerase-based reagent was inhibited by whole blood and amplification could
not be confirmed. On the other hand, Hot Start rTth (DNA) Kit was able to determine
SNPs from whole blood without purification.

[5] Related products

Product namePackage

Code No.

10,000 U

HSTTH-329

100 mL

QRZ-1B1250 mL

QRZ-1B2

1,000 mL

QRZ-1B4

5 mL

ROX-101<High efficient DNA Polymerase for PCR and RT-PCR>

Hot Start rTth DNA Polymerase<Reaction Buffer (containg Mg2+) for DNA amplification>

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

50x ROX reference dyeJAPANTOYOBO 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