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

Catalog #: EG-1061

$0.00

Product Description

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

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

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

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