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2024年6月30日发(作者：)
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F1787K
Hot Start TTx (DNA) Kit
HSTTX-101 250U
Store at -20°C
[1] Introduction
[2] Components
[3] Protocol
1. Standard reaction setup
2. Cycling conditions
[4] Example
[5] Related products
Contents
CAUTION
All 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.
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-5879-4900
/e/bio
********************
1
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
/e/bio
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 fast
cycle 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 for
real-time PCR using probe assays such as TaqMan
®
assay. This enzyme contains
neutralizing antibodies, thus allowing for Hot start PCR.
[1] Introduction
Features
-Excellent DNA Amplification Efficiency
The reaction composition is optimized based on TTx DNA Polymerase. TTx DNA
Polymerase has higher elongation capacity than general-purpose enzymes such as Taq
DNA Polymerase and Tth DNA Polymerase, TTx DNA Polymerase enable efficient PCR
even fast cycle condition.
[2] Components
- 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.
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) 1.25 mL x 2
Hot Start TTx DNA Polymerase (4U/ μL) 62.5 μL
Note:
-2x Reaction Buffer contains essential components for the reaction (buffer, salts, Mg
2+
,
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).
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-5879-4900
/e/bio
********************
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
2
/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 volume
Volume
X µL
10 µL
0.6 µL
0.6 µL
0.4 µL
0.25 µL
Y µL
20 µL
Final Concentration
1x
0.3 µM
0.3 µM
0.2 µM
1U
Notes:
-The recommended amount of primer should be 0.2-0.6 μM, and the amount of
TaqMan
®
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 too
much, it may cause non-specific reaction and detection sensitivity may be lowered.
2. Cycling conditions
Predenature :
Denature :
Annealing/extension :
The following cycle is recommended.
Temperature
95°C
95°C
60°C
Time
1 min.
15 sec.
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 from
the supplier of UNG.
- If sensitivity is not good, it may be improved by changing annealing/ extension
temperature between 55 ~ 65°C.
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO (SHANHAI) BIOTECH CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-5879-4900
/e/bio
********************
FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
3
/e/bio
Detection of african swine fever virus
African swine fever virus DNA was detected using TaqMan
®
probes. As a result of
performing 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.
[4] Example
[5] Related products
Product name
Package
10,000 U
100,000 U
100 mL
250 mL
1,000 mL
5 mL
Code No.
HSTTX-129
HSTTX-159
QRZ-1B1
QRZ-1B2
QRZ-1B4
ROX-101
Hot Start TTx DNA Polymerase
2+ ) for DNA amplification> 2x Buffer for rTth/ TTx (DNA) 50x ROX reference dye Hot Start rTth DNA Polymerase < Reaction Buffer (not containg Mg 2+ and Mg 2+ > 10,000 U 40 mL 400 mL 5mL HSTTH-329 QRT-1B1 QRT-1B2 QRT-MN1 5x Buffer for rTth/ Ttx (DNA/ RNA) 50 mM Mn (OAc) 2 25 mM MgCl 2 40 mL TAP-2S1 JAPAN CHINA TOYOBO CO., LTD. TOYOBO (SHANHAI) BIOTECH CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-5879-4900 /e/bio ******************** FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. 4
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