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

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

• 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

• 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:

2. Heat reaction for 5 minutes at 65°C. Spin briefly (5 sec) to pull down condensate and place immediately on ice.
3. Add 1 µL 10X M-MuLV RT Buffer and DNase-free water to a final volume of 10 µL per reaction.
4. Incubate:
   • If using Oligo (dT) or GSP primers: 2 minutes @ 42°C
   • If using Random primers: 2 minutes @ 25°C
5. 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).
6. Incubate at 42°C for 45-60 minutes.
7. Inactivate enzyme at 85°C for 10 minutes.
8. Store products at -20°C or proceed to next step.