Description
ZELLX® AMV Reverse Transcriptase (AMV RT) is a high-quality RNA-dependent DNA polymerase derived from Avian Myeloblastosis Virus (AMV) and optimized for first-strand cDNA synthesis, particularly from long, GC-rich, or highly structured RNA templates. Due to its superior thermal stability compared to M-MLV RT, AMV Reverse Transcriptase is well suited for applications requiring higher reaction temperatures.
AMV Reverse Transcriptase efficiently synthesizes complementary DNA (cDNA) from RNA templates using oligo(dT), random primers, or gene-specific primers. The enzyme performs optimally at 42–50 °C, which helps reduce RNA secondary structure and improves reverse transcription efficiency for difficult templates.
ZELLX® AMV Reverse Transcriptase exhibits multiple enzymatic activities, including RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities, providing robust and consistent cDNA synthesis. Although AMV RT has higher RNase H activity than M-MLV RT, this property can be advantageous in applications requiring controlled RNA degradation during cDNA synthesis.
Supplied at a concentration of 10 U/µL, ZELLX® AMV Reverse Transcriptase is provided as a complete solution, including a 5× Reaction Buffer for immediate use. The enzyme is purified to high homogeneity and is free of detectable RNase and DNase activities, including exonuclease and endonuclease contaminants, ensuring reliable and reproducible performance.
Manufactured under strict quality control standards, ZELLX® AMV Reverse Transcriptase delivers excellent stability and batch-to-batch consistency and integrates seamlessly into downstream workflows such as RT-PCR, RT-qPCR, cloning, and gene expression analysis.
Product Specifications:
Catalog Number: ZXB-06-209
Protocol:
First-Strand cDNA Synthesis Protocol Using AMV Reverse Transcriptase
To ensure optimal performance, the reaction is prepared as two separate mixes before combining.
Mix 1: RNA–Primer Preparation
- Prepare the RNA–primer mix
In an RNase-free tube, combine:
- 2 µg total RNA
(or 50–500 ng poly(A)+ RNA) - Reverse transcription primer
(use 0.5 µg primer per µg RNA) - Nuclease-free water to a final volume of 11 µL
Mix gently by vortexing and briefly centrifuge.
- Denature RNA
Incubate the mixture at 70 °C for 5 minutes to denature RNA secondary structures.
Immediately chill on ice.
Mix 2: Reverse Transcription Master Mix
- Prepare the RT master mix
In a separate tube, add:
- 5 µL 5× RT Buffer
- 2.5 µL dNTP mix (10 mM each)
- 20–40 U RNase inhibitor (optional; not provided)
- 2.5 µL 40 mM sodium pyrophosphate (prewarmed to 42 °C)
- 3 µL AMV Reverse Transcriptase (10 U/µL)
- Nuclease-free water to a final reaction volume of 25 µL
Mix gently and briefly centrifuge.
Reverse Transcription Reaction
- Combine Mix 1 and Mix 2
Add the contents of Mix 2 to Mix 1.
Mix gently by pipetting or brief vortexing and centrifuge briefly. - Incubate for cDNA synthesis
- 42 °C for 60 minutes when using oligo(dT) primers
- 37 °C for 60 minutes when using random hexamer primers
- Terminate the reaction
Heat at 70 °C for 10 minutes, then chill on ice. - Downstream use
The resulting cDNA can be used directly for PCR, qPCR, cloning, or other downstream applications, or stored at –20 °C.
