| 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. Unit Definition: One 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 37C. 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. Applications: Suitable for use in In Situ Transcription, cDNA Library Synthesis and Screening, mRNA Structure Determination and Ribosomal Pause Site Determination. Recommended Dilutions: M-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 1µg 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. Optimal dilutions to be determined by researcher. R1991A: 5X Reaction Buffer for Reverse Transcriptase, M-MLV (M-MLV-RT) 1x1ml (included): 5X: 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. 1X: 50mM Tris-HCl, pH 8.3 at RT, 75mM potassium chloride, 3mM magnesium chloride, 10mM DTT. Quality Control Assays Activity Assay: First-Strand cDNA Synthesis: First-strand cDNAs, of 1.2kb and 7.5kb control RNAs, are synthesized at 37C 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. Contaminant 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 37C, 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. Endonuclease 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 37C. 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). Storage and Stability: May be stored at 4C for short-term only. For long-term storage, store at -20C. Aliquots are stable for at least 6 months at -20C. 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. |
