ExcellScript Thermostable Reverse Transcriptase

ExcellScript Thermostable M-MuLV Reverse Transcriptase is a DNA polymerase which utilizes RNA as a substrate and exhibits no measurable proofreading 3′→5′ exonuclease function. This thermostable reverse transcriptase can perform cDNA synthesis reactions throughout a wide range of temperatures from 42°C to 72°C.

• First strand cDNA synthesis for RT-PCR and real-time RT-PCR
• Synthesis of cDNA for cloning and expression
• Generation of labeled cDNA probes for microarrays
• DNA labeling
• Analysis of RNA by primer extension
• Isothermal nucleic acid amplification such as LAMP

• The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
• Functionally tested in first strand cDNA synthesis.

1 unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37°C using poly r(A)/oligo (dT) as a substrate.

• Thermostable M-MuLV Reverse Transcriptase: 200 units/µL in 50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40, 50% glycerol, pH 7.6 @ 25°C
• 10X Reaction Buffer: 500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl₂, 100 mM DTT, pH 8.3 @ 25°C

1. Primer Annealing: Combine the following in an RNase-free reaction vessel:

1. Heat reaction for 5 minutes at 65°C. Spin briefly (5 sec) to pull down condensate and place immediately on ice.
2. Add 1 µL 10X M-MuLV RT Buffer and DNase-free water to a final volume of 10 µL per reaction.
3. Incubate:
   • If using Oligo (dT) or GSP primers: 2 minutes @ 42°C
   • If using Random primers: 2 minutes @ 25°C
4. Add 1 µL (200 units) thermostable M-MuLV Reverse Transcriptase and mix by gently pipetting sample. (Note: if using random primers, pre-incubate reaction @25°C for 10 minutes).
5. Incubate at 42 ~ 72 °C for 45-60 minutes.
6. Inactivate enzyme at 95°C for 10 minutes.
7. Store products at -20°C or proceed to next step.