Reverse Transcriptase, M-MLV (M-MLV-RT) Recombinant

Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is an RNA-dependent DNA polymerase that can be used in cDNA synthesis with long messenger RNA templates (> 5kb). M-MLV RT is the preferred reverse transcriptase for long mRNA templates because the RNase H activity of M-MLV RT is weaker than the commonly used Avian Myeloblastosis Virus (AMV) reverse transcriptase.\n\nUnit Definition: \nOne unit is defined as that amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37 degrees C. The reaction conditions are: 50mM Tris-HCl (pH 8.3), 7mM MgCl2, 40mM KCl, 10mM DTT, 0.1mg/ml BSA, 0.5mM [3H] dTTP, 0.025mM oligo(dT50), 0.25mM poly(A)400 and 0.01% NP-40.\n\nApplications:\nSuitable for use in In Situ Transcription, cDNA Library Synthesis and Screening, mRNA Structure Determination and Ribosomal Pause Site Determination.\n\nRecommended Dilutions:\nM-MLV Reverse Transcriptase is less processive than AMV Reverse Transcriptase, and therefore, more units of the M-MLV enzyme are required to generate the same amount of cDNA as in the AMV reaction. Thus, starting with 1microg of mRNA in a first-strand cDNA synthesis, 200 units of the M-MLV enzyme are recommended as opposed to 25 units of the AMV enzyme.\nOptimal dilutions to be determined by researcher.\n\nR1991A: 5X Reaction Buffer for Reverse Transcriptase, M-MLV (M-MLV-RT) 1x1ml (included):\n5X: Supplied as a liquid in 250mM Tris-Hcl, pH 8.3 at RT, 375mM potassium chloride, 15mM magnesium chloride, 50mM DTT. Dilute 1:5 prior to use.\n1X: 50mM Tris-HCl, pH 8.3 at RT, 75mM potassium chloride, 3mM magnesium chloride, 10mM DTT.\n\nQuality Control Assays\nActivity Assay: First-Strand cDNA Synthesis: First-strand cDNAs, of 1.2kb and 7.5kb control RNAs, are\nsynthesized at 37 degrees C for 1 hour using 200u of M-MLV Reverse Transcriptase, 1ug of each template, an oligo(dT)15 primer and radiolabeled dCTP. The minimum specification is the production of 120ng of first-strand cDNA. The cDNA product must be >90% full-length for the 1.2kb control RNA as observed by gel electrophoresis and autoradiography. For the 7.5kb control RNA, 25% of the cDNA must migrate at 7.5kb.\nContaminant Activity: DNase and RNase Assay: To test for nuclease activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 200u of M-MLV Reverse Transcriptase in 1X Reaction Buffer for one hour at 37 degrees C, and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for both DNase and RNase.\nEndonuclease Assay: To test for endonuclease activity, 1ug of Type I supercoiled plasmid DNA is incubated with 500u of M-MLV Reverse Transcriptase in 1X Reaction Buffer for one hour at 37 degrees C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting (analysis on 0.4ug of DNA).\n\nStorage and Stability: \nMay be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

R1991

Size

50KU

Form

Supplied as a liquid in 20mM Tris HCl, pH 7.5, 200mM sodium chloride, 0.1mM EDTA, 1mM DTT, 0.01% NP-40 and 50% glycerol.

Purity

(same/more than)90%, Verified by SDS-PAGE

References

US Biological Application Reference: 1. Cotttone, E. et al., J. Comp. Neurol. 485: 293-303 (2005). General References: 1. Houts, et al., J. Virology 29: 517-522 (1979). 2. Vrana, S.L., et al., Molecular Brain Research 34: 127-134 (1995). 3. Tecott, L., et al., Science 240: 1661-1664 (1988). 4. Van Gelder, R., et al., PNAS (USA) 87: 1663-1667 (1990). 5. Kievits, T., et al., J. Virol. Methods 35: 273-286 (1991). 6. Van Gemen, B., et al., J. Virol. Methods 49: 157-168 (1994). 7. Roth, M.J., et al., J. Biol. Chem. 260: 9326

Alternative Names

Moloney Murine Leukemia Virus; RNA-dependant DNA Polymerase