M-MLV Reverse Transcriptase

Description

Reliable Enzyme for First-Strand cDNA Synthesis

Product Description

ZELLX® M-MLV Reverse Transcriptase is a high-quality RNA-dependent DNA polymerase derived from Moloney Murine Leukemia Virus (M-MLV) and optimized for first-strand cDNA synthesis from RNA templates. This enzyme is widely used in molecular biology for reverse transcription applications requiring high sensitivity, reliability, and full-length cDNA synthesis.

M-MLV Reverse Transcriptase catalyzes the synthesis of complementary DNA (cDNA) from single-stranded RNA templates using oligo(dT), random primers, or gene-specific primers. Compared to more thermostable reverse transcriptases, M-MLV RT operates optimally at 37–42 °C, making it particularly suitable for structured or low-abundance RNA templates when gentle reaction conditions are preferred.

ZELLX® M-MLV Reverse Transcriptase exhibits low RNase H activity, which helps preserve RNA integrity during cDNA synthesis and supports the generation of longer cDNA products. The enzyme is purified to high homogeneity and is free of detectable DNase, RNase, and endonuclease contamination, ensuring reproducible and high-quality results.

Manufactured under strict quality control standards, ZELLX® M-MLV Reverse Transcriptase delivers consistent activity and batch-to-batch reproducibility. It is fully compatible with standard reverse transcription buffers and integrates seamlessly into downstream applications such as PCR, qPCR, and cloning workflows.

Product Specifications:

Catalog Number: ZXB-06-210

Protocol:

First-Strand cDNA Synthesis Protocol Using M-MLV Reverse Transcriptase

1. RNA–Primer Mix Preparation

In an RNase-free tube, combine the following components:

• Total RNA: 10 pg – 5 µg
  (or 10 pg – 0.5 µg mRNA / poly(A)+ RNA)
• Primer (choose one):
  • Oligo(dT)₁₂–₁₈ (50–60 µM):
    1 µL → 5–3 µM final concentration
  • Random hexamers (50–250 ng/µL):
    1 µL → 5–12.5 ng/µL final concentration
  • Gene-specific primer (2 µM):
    1 µL → 1 µM final concentration
• dNTP Mix (10 mM each):
  1 µL → 5 mM final concentration
• Nuclease-free water to a final volume of 16 µL

Mix gently by pipetting, briefly centrifuge, and keep on ice.

2. RNA Denaturation

Incubate the mixture at 65 °C for 5 minutes to denature RNA secondary structures.
Immediately chill on ice for at least 1 minute, then briefly centrifuge and return to ice.

3. Reverse Transcription Reaction Setup

Add the following components directly to the RNA–primer mix:

• 10× Reaction Buffer:
  2 µL
  (500 mM Tris-HCl, pH 8.3; 750 mM KCl; 30 mM MgCl₂; 100 mM DTT)
• RNase Inhibitor:
  1 µL (not provided)
• M-MLV Reverse Transcriptase (200 U/µL):
  1 µL (200 units)

Mix gently and briefly centrifuge.
Final reaction volume: 20 µL

4. Reverse Transcription

• Standard protocol:
  Incubate at 37 °C for 50 minutes
• When using random hexamer primers:
  • Incubate at 25 °C for 10 minutes
  • Then incubate at 37 °C for 50 minutes

5. Enzyme Inactivation

Terminate the reaction by heating at 70 °C for 15 minutes.

6. Downstream Use and Storage

The synthesized cDNA can be:

• Used immediately for PCR, qPCR, cloning, or other downstream applications, or
• Stored at –20 °C for later use.