Evolving Enzymes
Inovating IVD
TOROIVD® III Reverse Transcriptase is an engineered of M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase with removed RNase H activity to increase reaction efficiency thermal activity. The enzyme can be used to synthesize first-strand cDNA at temperatures up to 60°C, providing increased specificity, higher yields of cDNA, and more full length product than other reverse transcriptases. It is the preferred enzyme for applications requiring full-length cDNAs and high product yields from total RNA, mRNA, rRNA, etc.
DESCRIPTION
TOROIVD® III Reverse Transcriptase is an engineered of M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase with removed RNase H activity to increase reaction efficiency thermal activity. The enzyme can be used to synthesize first-strand cDNA at temperatures up to 60°C, providing increased specificity, higher yields of cDNA, and more full length product than other reverse transcriptases. It is the preferred enzyme for applications requiring full-length cDNAs and high product yields from total RNA, mRNA, rRNA, etc.
FEATURES
-RNase minus M-MLV RTase with improved performance.
-Exhibits excellent reaction efficiency at temperatures up to 60°C.
-Enables the synthesis of longer cDNAs than the WT-enzyme.
Components
RTM-002-2KU RTM-002-10KU RTM-002-100KU RTM-Buffer | TOROIVD® III Reverse Transcriptase, 2KU/20ul TOROIVD® III Reverse Transcriptase, 10KU/100ul TOROIVD® III Reverse Transcriptase, 100KU/1ml 5×RT Buffer 100ul/tube, 500ul/tube, 5ml/tube |
STORAGE BUFFER
20 mM Tris/HCl, 300 mM NaCl, 0.5% Triton X-100, 0.2M Trehalose, 5% (v/v) glycerol, pH 8.0
QUALITY CONTROL
Each batch of TOROIVD®III Reverse Transcriptase is confirmed
to contain <2% contaminating protein by SDS-PAGE. TOROIVD® III Reverse Transcriptase
are subjected to stringent quality control tests, are free of contaminating
exo-and endonuclease activity, and meet strict requirements with respect to DNA
contamination levels.
Protocol
1. First-Strand cDNA Synthesis
-Add the following components to a nuclease-free microcentrifuge tube:
Component | Volume |
Oligo(dT)20 (or Random primer) (or Gene specific primer) | 25pmoles (25pmoles) (5pmoles) |
10mM dNTPs | 2µL |
Template RNA | 2pg-5µg |
Nuclease-free water | Up to 10 µL |
-Heat the RNA/Primer mixture to 65°C for 5minutes and incubate on ice for at least 1 minute.
-Add the following components into the RNA/Primer mixture.
Component | Volume |
RNA/Primer mixture | 10 µL |
5*RT Buffer | 4µL |
TOROIVD® III Reverse Transcriptase | 1µL |
RNase inhibitor(40U/µL) | 1µL |
100mM DTT | 2µL |
Nuclease-free water | Up to 20 µL |
-Gently mix the reaction solutions and spin down in microcentrifuge.
-Perform Reverse transcription with the following protocol:
42ºC 20-60min
99ºC 5min
2. 1-Step RT-qPCR
Step 1: Prepare the reaction mixture
-Ensure that all reagents are properly thawed and
mixed.
-Prepare a PCR master mix containing the appropriate volume of all reaction
components common to all or a subset of reactions to be performed.
-Calculate the required volumes of each component based on the following table:
Component | 20 µL reaction | Final conc. |
PCR-grade water | Up to 20 µL | N/A |
2×5G qPCR Buffer GB | 10.0 µL | 1× |
TOROIVD® 5G DNA polymerase | 0.25 µL | 1U |
TOROIVD®III Reverse transcriptase | 0.3µL | 30U |
Recombinant RNase inhibitor | 0.25µL | 20U |
10 μM Forward Primer | 1µL | 0.5μM |
10 μM Reverse Primer | 1µL | 0.5 μM |
10µM Taqman Probe | 0.4µL | 0.2μM |
Template RNA | As required | As required |
-Gently mix the reaction solutions and spin down in microcentrifuge.
Step 2: Run the RT-qPCR
-Perform PCR with the following cycling protocol:
Step | Temperature | Duration | Cycles |
Reverse transcription | 52ºC | 5min | 1 |
Initial denaturation | 95 ºC | 1-5min | 1 |
Denaturation | 95ºC | 10 sec | 40cycles |
Annealing/ Extension | 60ºC | 20sec |
Notes: The indicated RT temperature of TOROIVD®III Reverse transcriptase can be optimized between 50-60°C, and time between 5-15min.
THERMAL STABILITY
TOROIVD® III Reverse transcriptase maintained all the performance at 37°C for one week.
STORAGE
This reagent can be stored at 4°C for 1 months. For longer storage,
this reagent should be kept at -20°C.
