This is a one-step qRT-PCR reagent using our unique enzyme TTx DNA Polymerase. TTx DNA Polymerase has reverse transcriptase activity, and this activity is strongly promoted in the presence of Mn2+. This activity can be used to perform reverse-transcription reactions and PCR with the same enzyme. Because TTx DNA Polymerase has reverse transcriptase activity, it can amplify with high efficiency from both DNA and RNA. TTx DNA Polymerase also has 5'→3' exonuclease activity, so it can be used for real-time PCR, for example using TaqMan® probes. This reagent contains a mixture of neutralizing antibodies, allowing for highly specific Hot Start PCR.
- Highly efficient one-enzyme system and 1-step qRT-PCR
TTx DNA Polymerase has high reverse transcriptase activity, allowing efficient qRT-PCR even with small quantities of template. This reagent can also be used to detect DNA.
- Detectability with high-speed cycles
Taking advantage of the high amplification efficiency, TTx DNA Polymerase allows efficient amplification even with high-speed cycles.
- Reverse transcription is possible at high temperatures
The reverse-transcription reaction is possible at 60℃, which makes this reagent suitable for amplifying targets that are GC-rich or that easily adopt higher-order structures.
TaqMan® assays or hybridization probe assays using RNA template
TaqMan® assays or hybridization probe assays using DNA template
Store at -20℃
This kit includes the following components for 250 reactions, 20 μL total reaction volume. All reagents should be stored at -20°C.
|5x Buffer for rTth/ TTx (DNA/ RNA)||1 mL|
|2 mM dNTPs||1 mL|
|50 mM Mn(OAc)2||250 μL|
|Hot Start TTx DNA Polymerase (4U/ μL)||62.5 μL|
5× Buffer for rTth/TTx (DNA/RNA) is 5× RT-PCR buffer not containing Mn2+ or dNTPs. Add template DNA, primers, 2 mM dNTPs, 50 mM Mn(OAc)2, Hot Start TTx DNA Polymerase, and adjust to 1× concentration with sterile DNase- and RNase-free water.
Hot Start TTx DNA Polymerase is a mixture of TTx DNA Polymerase and Hot Start antibodies.
This kit does not contain a passive reference dye (ROX). When using a passive reference dye to compensate for fluorescence intensity and dispensing error between wells, please use the separately sold 50× ROX reference dye (Code No. ROX-101).
Example 1.Detection of influenza virus RNA
Influenza virus RNAs were detected using the Hot Start TTx (RNA) Kit and Tth DNA Polymerase-based real-time PCR reagent. When 1 µL diluted, purified RNA was included in the 20µL reaction solution, the Hot Start TTx (RNA) Kit was more sensitive than the Tth DNA Polymerase-based reagent.
Example 2.High-speed cycling one-step qRT-PCR
The Hot Start TTx (RNA) Kit and products from other companies were used to detect influenza virus RNAs using TaqMan® probes and high-speed cycles. Only the Hot Start TTx (RNA) Kit resulted in efficient amplification.