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AMV Reverse Transcriptase

#EP0641 Supplied with: 5X AMV RT Buffer	1000 u (20 u/µl) 1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0641 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Temperature optimum of activity at 45-50°C.
• Highly thermostable, can be used at temperatures up to 60°C.
• Suitable for synthesis of full-length cDNA as long as 13 kb.
• Incorporates modified nucleotides.

Description
AMV Reverse Transcriptase (RT) is a recombinant Avian Myeloblastosis Virus reverse transcriptase expressed in E.coli. AMV RT is a heterodimer composed of nonidentical subunits alfa and beta. It possesses multiple enzymatic activities including RNA- and DNA-directed DNA polymerase, DNA-RNA unwinding activity, a sequence-specific Mn²⁺-dependent endonuclease and ribonuclease H.

Source
E.coli cells with a cloned pol gene of AMV.

Applications

• First strand cDNA synthesis, see protocol.
• RT-PCR.
• Synthesis of cDNA for cloning.
• RT-PCR of GC-rich templates and templates with high degree of secondary structure.
• Synthesis of labeled cDNA probes.
• Analysis of RNA by primer extension (1).

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested in first strand cDNA synthesis.

Concentration
20 u/µl

Definition of Activity Unit
One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 37°C.
Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 8 mM MgCl₂, 30 mM KCl, 1 mM DTT, 0.5 mM dTTP, 0.4 MBq/ml [³H]-dTTP, 0.4 mM polyA•oligo(dT)_12-18.

Storage Buffer
The enzyme is supplied in: 200 mM potassium phosphate, 2 mM DTT, 0.2% (v/v) Triton X-100 and 50% (v/v) glycerol (pH 7.2).

5X AMV RT Buffer
250 mM Tris-HCl (pH 8.5 at 25°C), 40 mM MgCl₂, 150 mM KCl, 5 mM DTT.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate. Pyrophosphate (Na-PP_i) at a 0.5 mM concentration reduces AMV RT activity to 50%. Na-PP_i at a <4 mM concentration increases the yield of full-length cDNA significantly (2).
• Inactivated by heating at 85°C for 5 min.

Related Products

Primers dNTP Mix, 10 mM each dNTP Mix, 25 mM each RiboLock™ RNase Inhibitor Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP Taq DNA Polymerase (recombinant) Taq DNA Polymerase (native): without BSA with BSA Pfu DNA Polymerase: native recombinant 2X PCR Master Mix Hot Start Taq DNA Polymerase TrueStart™ Taq DNA Polymerase PyroStart™ Fast PCR Master Mix (2X) High Fidelity PCR Enzyme Mix Long PCR Enzyme Mix DNase I, RNase-free DNA Polymerase I, E.coli RNase H, E.coli DEPC-treated Water Water, nuclease-free

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.

Updated vasario 05, 2008 15:26