Description
EnzymoPure™ M-MuLV Reverse Transcriptase is a modified and optimized Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase. It is a DNA polymerase that uses RNA or DNA as template to synthesize complementary DNA strands in the presence of primers. It also has Ribonuclease H (RNase H) activity, which can specifically degrade the RNA in RNA-DNA hybrids, but not single-stranded RNA or double-stranded RNA.FeaturesApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling; RNA analysis by primer extension.Source：Recombinant protein expressed in E. coli. The RT M-MuLV reverse transcriptase is encoded by the mutation-optimized pol gene encoding M-MuLV reverse transcriptase.Enzyme Activity: One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction in 10 min at 37℃. Enzyme activity is assayed in 50 mM Tris-HCl (pH 8.3), 6 mM MgCl2, 10 mM DTT, 40 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml [3H]-dTTP, 0.4 mM polyA•oligo(dT)12-18.Purity: Free of DNA endonuclease, DNA exonuclease, phosphatase, and RNase other than the RNase H enzyme activity contained in the RTTM M-MuLV Reverse Transcriptase.Enzyme storage buffer：50 mM Tris, pH 8.3, 100mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% Triton X-100 and 50% glycerol.Inactivation or inhibition：M-MuLV Reverse Transcriptase can be inactivated by incubation at 70℃ for 10 minutes, or inhibited by chelating agents including EDTA and EGTA, inorganic phosphate, pyrophosphate, and polyamine.This product is sufficient for 10 reverse transcription reactions when used in a reaction volume of 20µl.Precautions：Please refer to the instructions for reverse transcription of RNAs with high GC content.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1.First-strand cDNA Synthesis:a. Set up the reaction in a nuclease-free PCR tube as follows:RNA Template (one of the three types of RNA)Total RNA0.1-5µgPoly(A) RNA/mRNA10-500ngSpecific RNA0.01pg-500ngPrimer (one of the three types of primers)Oligo(dT)180.5µg (or100pmol)random hexamer0.2µg (or100pmol)Gene specific primer15-25pmol(optional) For RNAs with high GC content or complex secondary structures, incubate the mixture of primer and template at 65℃ for 5 minutes, and immediately put it on ice to disrupt RNA secondary structures.DEPC-treated Water-To 13.7µl *Reaction Buffer (5X)-4µlRNase Inhibitor-0.5µl **dNTP Mix (25 mM each)-0.8µl ***RTTM M-MuLV Reverse Transcriptase-1µlTotal Volume-20µl* ‘To 13.7µl' means filling the mixture of template and primer to a total volume of 13.7µl with DEPC-treated Water. ** The volume of RNase Inhibitor may vary depending on the type of RNase Inhibitor used. If the volume of RNase Inhibitor is not 0.5µl, adjust the volume of DEPC-treated Water accordingly.*** The volume of dNTP mix varies depending on the concentration of dNTP stock. If the volume of dNTP is not 0.8µl, adjust the volume of DEPC-treated Water accordingly.b. Mix well by vortex or pipetting gently, centrifuge briefly to collect liquid at the bottom of PCR tube.c. If using Oligo(dT)18 or gene-specific primers, incubate the reaction mixture at 42℃ for 60 minutes. If random hexamer is used, incubate at 25℃ for 10 minutes followed by 60 minutes at 42℃. Note: For RNA templates with high GC content, incubate the reaction at 45℃ for 60 minutes.d. Incubate at 70℃ for 10 minutes to stop the reaction. Note: Heat-inactivation of reverse transcriptase is not recommended for long cDNA over 5kb, as this method may cause shearing of long cDNA fragments. In such a case, phenol-chloroform extraction or column purification can be considered.e. The reverse transcription products can be used directly for subsequent experiments such as PCR, or stored at -20℃ for future use. We recommend using 2µl reverse transcription products in a PCR reaction volume of 50µl.For other uses, please refer to the relevant literature of M-MuLV reverse transcriptase.FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomenon, because the amount of RNA template is low, and the amount of reverse transcription products in different size is even lower.2. No specific product can be amplified from the reverse transcription product.a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. Reference genes can be amplified but not the target gene, indicating primers of target gene are not well designed or the expression of the target gene is too low to be detected. b. Inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well designed