Reverse Transcriptase with Stability at Higher Temperatures
Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) catalyzes DNA polymerization using template DNA, RNA or RNA:DNA hybrids. It requires a primer (DNA primers are more efficient than RNA primers) as well as Mg2+ or Mn2+. The enzyme possesses an intrinsic RNase H activity. Both nonionic detergents and sulfhydryl compounds stabilize the enzyme activity in vitro.
Features
- High Concentration: Cat.# M9004 contains 600 units of AMV RT at 20–25u/μl.
- 5X Reaction Buffer: 250mM Tris-HCl (pH 8.3 at 25°C), 250mM KCl, 50mM MgCl2, 2.5mM spermidine, 50mM DTT.
- Temperature Stability: AMV RT is the preferred reverse transcriptase for templates with high secondary structure due to its stability at higher reaction temperatures (37–58°C).
Applications
- First- and second-strand synthesis of cDNA.
- Primer extensions and RNA sequencing.
- RT-PCR. Up to 10μl of an RT reaction containing AMV RT and the supplied AMV RT Reaction Buffer can be added to a 50μl PCR amplification reaction that uses Taq DNA polymerase. If GoTaq® DNA Polymerase or PCR Master Mix are used, up to 25μl of an RT reaction can be added per 50μl PCR.
Further Reading
- Kacian, D.L. (1977) Meth. Virol. 6, 143.
- Mierendorf, R.C. and Pfeffer, D. (1987) Meth. Enzymol. 152, 563–6.
