Description
First-Strand cDNA Synthesis with Long mRNA Templates
Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RT [H–]), is an RNA-dependent DNA polymerase that can be used in cDNA synthesis with long messenger RNA templates (>5kb). This form of M-MLV Reverse Transcriptase has been genetically altered to remove the associated RNase H activity. Although many researchers are successful in using M-MLV RT (H+) for analytical and some preparative cDNA applications, reverse transcriptases lacking RNase H activity provide another option to prepare long cDNAs and libraries containing a high percentage of full-length cDNA.
Advantages
RNase H Minus: Provides optimal conditions to prepare full-length cDNA from long RNA templates.
Provided with 5X Reaction Buffer: 250mM Tris-HCl (pH 8.3 at 25°C), 375mM KCl, 15mM MgCl 2 , 50mM DTT.
May Be Heat-Inactivated: M-MLV RT is inactivated by heating at 70°C for 10 minutes.
Applications
First-strand cDNA synthesis.
Primer extension.
Technical Details
Storage Buffer: 20mM Tris-HCl (pH 7.5 at 25°C), 0.2M NaCl, 0.1mM EDTA, 1mM DTT, 0.01% Nonidet® P-40 and 50% glycerol.
Source: Recombinant E. coli strain.
QC Tests: Activity, SDS-PAGE/purity, DNase, RNase, endonuclease.
Unit Definition: One unit is the amount of enzyme required to incorporate 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37°C. The reaction conditions are: 50mM Tris-HCl (pH 8.3 at 25°C), 7mM MgCl 2 , 40mM KCl, 10mM DTT, 0.1mg/ml BSA, 0.5mM [ 3 H]dTTP, 0.025mM oligo(dT), 0.25mM poly(A) and 0.01% NP-40.
