Evolving Enzymes
Inovating IVD
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.
