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ExcellScript M-MLV (MMLV) Reverse Transcriptase RNase H minus

Cat #: EG-1061

   
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Description

ExcellScript M-MuLV Reverse Transcriptase RNase H minus is an RNA-dependent DNA polymerase with no detectable RNase H activity. ExcellScript can be used to generate first-strand cDNA from polyA mRNA or total RNA for use in downstream applications such as RT- PCR, cDNA cloning or library construction for RNA-Seq. Point mutations in the RNase H domain increase the thermostability of the enzyme and support greater cDNA yield of full-length transcripts than wild type M-MLV Reverse Transcriptase
Applications
  • 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
Source A recombinant E. coli strain carrying an engineered Moloney-Murine Leukemia Virus Reverse Transcriptase gene
Unit Definition 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.
Components
  • 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
Storage Condition -20 °C
Protocol
  1. Primer Annealing: Combine the following in an RNase-free reaction vessel:
Amount Description Final Concentration
1 µL 25mM dNTP Solution (N2050L 2.0 mM
X µL 1ng-2µg Total RNA -or-  
X µL 5-500 ng mRNA (polyA selected)  
1 µL Oligo (dT)12-18 (500 µg/ml) -or- 40 µg/mL
1 µL Random Primers (125 µg/ml) -or 10 µg/mL
1 µL GSP Primer (2 pmol) 165 µM
X µL Sterile Water N/A
10 µL Total Volume
  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) 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°C for 45-60 minutes.
  6. Inactivate enzyme at 85°C for 10 minutes.
  7. Store products at -20°C or proceed to next step.