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RevertAid Premium Reverse Transcriptase

Not available in US and Canada

An advanced RT enzyme lacking RNase H activity developed through in vitro evolution of M-MuLV RT.


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Description
Specifications
Supporting Data
References
Resources

Thermo Scientific RevertAid Premium Reverse Transcriptase was developed through in vitro evolution of M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity, but lacks RNase H activity. The engineered enzyme features dramatically improved thermostability, robustness, and an increased synthesis rate compared to wild type M-MuLV RT.

The eliminated RNase H activity enables the enzyme to produce very long RNA transcripts up to 20 kb. Due to its high thermostability, the enzyme maintains full activity during the entire reverse transcription reaction and generates high yields of cDNA. The reaction temperature can be increased up to 60°C for efficient transcription of RNA regions with a high secondary structure, or to improve specificity using gene-specific primers.

Highlights

Thermostable – 90% active after incubation at 50°C for 60 minutes in a reaction mixture
Active up to 60°C
High yields of full-length cDNA as long as 20 kb
Efficient – complete cDNA synthesis in 30 minutes
Incorporates modified nucleotides

Applications

First strand cDNA synthesis for RT-PCR and RT-qPCR
Reverse transcription at elevated temperatures to reduce effects of secondary structure
Synthesis of cDNA for cloning and expression
Generation of labeled cDNA probes for microarrays
DNA labeling
Analysis of RNA by primer extension

Includes

RevertAid Premium Reverse Transcriptase (200 U/µL)
5X RT Buffer


Storage Condition	-20 C
Hazardous	No
5x RT Buffer	250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, 15 mM MgCl₂, 50 mM DTT
Definition of Activity Unit	One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 minutes at 37°C. Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 4 mM MgCl₂, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/mL [3H]-dTTP, 0.4 mM polyA oligo(dT)_12-18.
Inactivation	Inactivated by heating at 85°C for 5 minutes
Inhibition	Metal chelators, inorganic phosphate, pyrophosphate and polyamines.
Quality Control	The absence of endodeoxyribonucleases, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests. Functionally tested in first strand cDNA synthesis.
Source	E. coli cells carrying an engineered pol gene fragment of Moloney Murine Leukemia Virus.
Storage Buffer	50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100 and 50% (v/v) glycerol.

High thermostability of RevertAid Premium Reverse Transcriptase at 50°C

Figure 1 | RevertAid Premium Reverse Transcriptase retains more activity over time. Reverse transcriptases were incubated in 1X reaction buffer. At the indicated time points (5 to 240 minutes), enzyme activity was determined in a standard activity assay.

Synthesis of long transcripts up to 20 kb using RevertAid Premium Reverse Transcriptase

Figure 2 | RevertAid Premium Reverse Transcriptase synthesizes transcripts up to 20 kb long. 1 µg of total mouse tongue RNA and oligo(dT)₁₈ primer were used in a 60 minute reverse transcription reaction at 50°C with RevertAid Premium Reverse Transcriptase. Synthesized cDNA was used as a template in subsequent PCR with the Long PCR Enzyme Mix and primers specific to different regions of the nebulin gene.

Accurate quantification across 8 orders of magnitude with the RevertAid Premium Reverse Transcriptase

Figure 3 | RevertAid Premium Reverse Transcriptase enables accurate quantification of transcripts over 8 orders of magnitude. RevertAid Premium Reverse Transcriptase was used in parallel first strand synthesis reactions with serial dilutions of in vitro transcribed GAPDH RNA (10-10⁸ copies) for 15 minutes at 50°C. The synthesized cDNA was used in qPCR with Maxima SYBR Green/ROX qPCR Master Mix and primers specific to the GAPDH gene. Reaction efficiency was 96%, slope -3.43, R² = 0.999.