AZscript™ Reverse Transcriptase
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.
Reduced RNase H activity
AZscript Reverse Transcriptase shows excellent performance and sensitivity in RT-qPCR
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).
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.
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.
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.
Additional Data and Information
We are pleased to provide additional data relating to AZscript RT upon request.
We appreciate suggestions for product modifications.
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