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RevertAid™ H Minus M-MuLV Reverse Transcriptase
Not available in the USA

#EP0451 Supplied with: 5X Reaction Buffer	10,000 u (200 u/µl) 1 ml
#EP0452 Supplied with: 5X Reaction Buffer	5x10,000 u (200 u/µl) 5x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0451, #EP0452 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Generates high yields of full-length cDNA as long as 13 kb, see picture below.
• Temperature optimum of activity at 42-45°C.
• Active up to 55°C.
• Incorporates modified nucleotides (e.g. Cy3-, Cy5-, rhodamine-, aminoallyl-, fluorescein-labeled nucleotides).

Description
The RevertAid™ H Minus M-MuLV Reverse Transcriptase (RT) is a genetically modified M-MuLV RT. It differs from the M-MuLV RT by its structure and catalytic properties. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity, but lacks ribonuclease H activity specific to RNA in RNA-DNA hybrids. RNase H activity is eliminated by a point mutation in the RNase H domain of M-MuLV RT (1, 2).

Source
E.coli cells carrying a cloned fragment of the mutated pol gene encoding Moloney Murine Leukemia Virus reverse transcriptase.

Molecular Weight
76.1 kDa monomer.

Applications

• Generation of first strand cDNA (3) for use in:
  • PCR, see Protocol for First-strand cDNA Synthesis;
  • real-time PCR (4, 5);
  • second strand cDNA synthesis.
• Generation of probes for microarrays (6).
• DNA labeling (3).
• Analysis of RNA by primer extension (3).

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

Concentration
200 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), 6 mM MgCl₂, 10 mM DTT, 40 mM KCl, 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: 50 mM Tris-HCl (pH 8.3), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100 and 50% (v/v) glycerol.

5X Reaction Buffer
250 mM Tris-HCl (pH 8.3 at 25°C), 250 mM KCl, 20 mM MgCl₂, 50 mM DTT.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate, pyrophosphate and polyamines (2).
• Inactivated by heating at 70°C for 10 min.

Synthesis of long cDNA using RevertAid™ H Minus M-MuLV Reverse Transcriptase.
A 1 µg of total mouse heart RNA and oligo(dT)₁₈ primer were used in reverse transcription reaction with the RevertAid™ H Minus M-MuLV Reverse Transcriptase. Synthesized cDNA was used as a template in subsequent PCR with Long PCR Enzyme Mix (#K0181). A PCR primer specific to the 5’-end of mouse dystrophin gene mRNA and a series of flanking primers were used to amplify different regions of the gene.
M₁ - GeneRuler™ DNA Ladder Mix
1-6 - RT-PCR products
M₂ - Lambda - pUC Mix Marker

Related Products

dNTP Mix, 10 mM each RiboLock™ RNase Inhibitor RevertAid™ First Strand cDNA Synthesis Kit Primers Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP Taq DNA Polymerase (recombinant) Taq DNA Polymerase (native): without BSA with BSA 2X PCR Master Mix High Fidelity PCR Enzyme Mix Long PCR Enzyme Mix DNase I, RNase-free DNA Polymerase I, E.coli RNase H, E.coli T4 DNA Ligase Pyrophosphatase, Inorganic (from yeast) DNA ladders RiboRuler™ RNA ladders DEPC-treated Water

References

1. Verma, I.M., Reverse transcriptase, The Enzymes (Boyer, P.D., ed), Academic Press Inc., 14, 87-103, 1981.

2. Gerard, G.F., D’Alessio, J.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.
3. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
4. Schmidt, A., Su, Y.H., et al., UPS1 and UPS2 from Arabidopsis Mediate High Affinity Transport of Uracil and 5-Fluorouracil, J. Biol. Chem., 279, 44817-44824, 2004.
5. Papavinasasundaram, K.G., et al., Deletion of the Mycobacterium tuberculosis pknH Gene Confers a Higher Bacillary Load during the Chronic Phase of Infection in BALB/c Mice, J. Bacteriol, 187, 5751-5760, 2005.
6. Turk, R., et al., Gene expression variation between mouse inbred strains, BMC Genomics, 5:57, 2004.

Updated kovo 18, 2008 11:27