AZscriptᵀᴹ Reverse Transcriptase

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AZscript™ Reverse Transcriptase (RT) is an RNA-dependent DNA polymerase used to synthesize complementary DNA (cDNA) from an RNA template.

While originating from Moloney Murine Leukemia Virus (M-MLV), AZscript RT has been engineered for increased thermostability and reduced RNase H activity.

In cDNA synthesis a higher reaction temperature can reduce RNA secondary structures and improve efficiency and specificity of the reaction.

Furthermore, a reduction in RNase H activity increases performance of the cDNA synthesis, especially for longer transcripts.

The combination of these tailored features ensures that AZscript Reverse Transcriptase provides an effective and reliable solution for various cDNA synthesis applications.

Key Features

Increased thermostability.
Reduced RNase H activity.

Applications

cDNA synthesis
Demonstrated performance in RT-qPCR

Figures

Figure 1. AZscript shows excellent performance and sensitivity in RT-qPCR

A serial dilution of MS2 Armored RNA (Asuragen, 52000) was heat lysed (75℃, 5min) and used as template for cDNA synthesis using gene-specific primers, according to manufacturer’s instructions. A volume equivalent to 2 μl of the cDNA was amplified in a 25 μl reaction using TaqPath™ qPCR Master Mix, CG. Data showing amplification plots (A) and standard curve (B) for the dilution series corresponding to 100 000 down to 10 copies of RNA, in triplicate measurements.

Figure 2. AZscript shows equal or better performance compared to a commonly used reverse transcriptase

A serial dilution of MS2 Armored RNA (Asuragen, 52000) was heat lysed (75℃, 5min) and used as template for cDNA synthesis using gene-specific primers, according to manufacturer’s instructions (ArcticZymes and Vendor A). A volume equivalent to 2 μl of the cDNA was amplified in a 20 μl reaction using TaqPath™ qPCR Master Mix, CG. Data showing average quantification cycle (Cq) for the dilution series corresponding to 100 000 down to 10 copies of RNA, in triplicate measurements. Error bars represents the standard deviation (n=3). *Reverse transcriptase from Vendor A failed to detect 10 copies (Cq set to 40).

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Properties

Source

Recombinantly produced in E. Coli

Size

79.5 kDa

Unit Definition

One unit is defined as the amount of enzyme needed to incorporate 1 nmol of dTTP into acid-precipitable material in 10 min at 37°C using poly(A)⋅oligo(dT)¹⁵ as template-primer. The enzyme is assayed in 37.5 mM Tris-HCl pH 8.3 @ 25°C, 40 mM KCl, 6 mM MgCl₂, 0.75 mg/ml Poly(A), 0.03 mM oligo(dT)₁₅, 10 mM DTT, 0.5 mM dTTP, 0.025 mCi/ml 3H-dTTP, 0.1 mg/ml BSA, 0.02% Triton X-100.

Temperature Optimum (>80%)

38-47°C

Inactivation

70°C, 15 minutes

Suggested 5X Reaction Buffer

250 mM Tris-HCl pH 8.3 (25°C), 375 mM KCl, 15 mM MgCl₂

Storage and Stability

The enzyme is stable at -20°C for up to 9 months in the supplied storage buffer. Keep AZscript RT on ice while handling. Additional data on stability is available on request.

Quality Control

ArcticZymes is dedicated to the quality of our products. AZscript RT is manufactured at our ISO 13485 certified facility in Norway.

dsDNA Endonuclease Activity

200 U AZscript RT was incubated with a supercoiled plasmid (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any transformation of closed circular DNA to nicked DNA.

ssDNA Endonuclease Activity

200 U AZscript RT was incubated with M13 ssDNA (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of ssDNA degradation.

ssDNA and dsDNA Exonuclease Activity

200 U AZscript RT was incubated with either 3H-dATP labelled ds or ssDNA (0.5 µg, 500 bp) for 4 hours at 37°C. Acid soluble radioactivity from labelled DNA was not significantly over blank test for either substrate.

RNase Activity

200 U AZscript RT was incubated with a 2 kb RNA transcript (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of RNA degradation.

Recommended Protocols

Ordering Info

Quantity

Product Name

SKU

Pack Size

Concentration

Price

T7 RNA Polymerase

74100-201

10 000 U

50 U/µl

$ 145.00 USD

AZscriptᵀᴹ Reverse Transcriptase