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TOROIVD® III Reverse Transcriptase
    Publish time 2020-08-07 15:04    

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 Transcriptase2KU/20ul

TOROIVD® III Reverse Transcriptase10KU/100ul

TOROIVD® III Reverse Transcriptase100KU/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 

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°Cand 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.





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