AMV Reverse Transcriptase
Used for synthesizing cDNA for PCR, cloning and hybridization probes
Purified virion of Avian myeloblastosis Virus.
AMV Reverse Transcriptase catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids(1). It consists of two polypeptide chains, one of which contains a 5'→3' polymerase activity and the other an RNase H activity. Reverse transcriptase requires Mg2+ or Mn2+ and is used for first strand synthesis of complementary DNA (cDNA) from mRNA template(2). Under proper conditions, high yields of full-length cDNA can be obtained with AMV Reverse Transcriptase(3). AMV Reverse Transcriptase is also useful in the amplification of RNA(4).
Molecular Weight: 157 kDa
Inactivation: 75° for 10 min or by adding 2 μl of 0.5M EDTA for a 50 μl reaction.
Tested for contaminating endonucleases, exonucleases and ribonucleases.
200mM potassium phosphate (pH 7.2), 2mM DTT,0.2% Triton X-100, 50% glycerol.
The reaction mixture (100 μl) contains 50mM Tris-HCI (pH 8.3), 6mM MgCl2, 40mM KCI, 0.5mM radiolabeled TTP, 0.4mM Poly (rA)n:oligo(dT)50. Incubation is at 37°C for10 min.
One unit is the amount of enzyme required to incorporate1 nmol of radiolabeled nucleotide into acid insoluble product in 10 min at 37°C.
Tested User Friendly™ Functional Test:
RT-PCR amplification of targets from total RNA.
Functionally Tested 5X AMV RT Reaction Buffer (1 ml included, PN 76218):
250mM Tris-HCl (pH 8.3), 40mM MgCl2, 250mM NaCl, 5mM DTT
- KACIAN, D. L. (1977) in Methods in Virology, eds.K. Maramorosch and H. Koprowski 6, 143-184.
- GOODMAN, H. M. AND MACDONALD, R. J. (1979) Methods Enzymol. 68, 75-90.
- BERGER, S. L., WALLACE, D. M., PUSKAS, R. S. AND ESCHENFELDT, W. H. (1983) Biochemistry 22, 2365-2372.
- PCR Protocols: A Guide to Methods and Applications (1990) eds. M. A. Innis, D. H. Gelfand and John J. Sni, (Academic Press, New York). p. 23.
- Synthesizing cDNA for PCR, cloning and hybridization probes.
- Filling-in and labeling the 3' termini of DNA with 5' protruding ends.
- RNA sequencing.
- Amplification of RNA(4).
- Primer extension assays.
Shipped on dry ice. Store at -20°C. Avoid freeze-thaw cycles, which result in loss of enzyme activity.