DESCRIPTION
Hot Start TTx (DNA) Kit is qPCR 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.
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.
- Detectability with high-speed cycles
Taking advantage of the high amplification efficiency, TTx DNA Polymerase allows efficient amplification even with high-speed cycles.
- 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.
- Allows multiplex PCR
By using TaqMan® probes with different detection wavelengths, it is possible to detect more than one target simultaneously. You can detect control genes and target genes within the same reaction, allowing rapid, simple, and accurate gene quantification.
- 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.Uracil-DNA Glycosylase (UNG), Heat-labile(Code No. UNG-101) can be used.
APPLICATIONS
TaqMan® assays or hybridization probe assays using DNA template
STORAGE CONDITION
Store at -20℃
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) | 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, 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).
APPLICATION DATA
Example 1.Detection of Bordetella pertussis IS481 gene and human G6PDH gene.
Amplifications of the IS481 gene using B. pertussis DNA (150,000, 3,000, 600, 120, 24, 5, and 1 copies) or the G6PDH gene using 5-fold dilutions (four steps) of cDNA from HeLa cell total RNA were performed using real-time PCR reagents (Hot Start TTx or Taq DNA Polymerase). We obtained efficient amplification of all targets using the Hot Start TTx kit.
Example 2.Real-time PCR with high-speed cycles
Amplifications of African swine fever viral DNA were performed using real-time PCR reagents (Hot Start TTx or Taq DNA Polymerase) with high-speed cycles. Only the Hot Start TTx (DNA) Kit showed efficient amplification in fast-cycling conditions.
Example 3.Effect of adding blood plasma to reactions
Amplifications of African swine fever viral DNA were performed using real-time PCR reagents (Hot Start TTx or Taq DNA Polymerase). Plasma (2.5 µL) was included in the 20µL reaction solution. The Taq DNA Polymerase-based reagents were inhibited by the plasma and no amplification was observed. However, amplification occurred with TTx DNA Polymerase, without inhibition.
Example 4.Detection of enterobacteria by multiplex PCR
Amplifications of four target genes* in one reaction were performed using real-time PCR reagents (Hot Start TTx or Taq DNA Polymerase). The target genes were difficult to detect with the Taq DNA Polymerase-based reagents, whereas with the Hot Start TTx (DNA) Kit, all four targets were detectable.
*The target genes were derived from Salmonella, Shigella and Escherichia coli O157 plus an internal control(IC) gene.
