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RevertAid H Minus Reverse Transcriptase


A recombinant M-MuLV RT lacking RNase H activity for high yield cDNA synthesis.
  
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Thermo Scientific RevertAid H Minus Reverse Transcriptase is a recombinant M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity, but lacks RNase H activity due to a point mutation in the RNase H domain (see References 1, 2). RevertAid H Minus Reverse Transcriptase does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cDNA, and therefore high yields of full-length cDNA from long templates are obtained. RevertAid H Minus Reverse Transcriptase maintains activity over a wide temperature range (42 to 50°C).

Highlights

  • High yields of full-length first strand cDNA up to 13 kb
  • Optimum activity at 42 to 45°C
  • Active up to 55°C
  • Incorporates modified nucleotides (e.g., Cy3-, Cy5-, rhodamine-, aminoallyl-, fluorescein-labeled nucleotides)

Applications

  • First strand cDNA synthesis for RT-PCR and real-time RT-PCR (see Reference 3)
  • Reverse transcription at elevated temperatures to reduce effects of secondary structure
  • Synthesis of cDNA for cloning and expression
  • Generation of labeled cDNA probes for microarrays
  • DNA labeling (see​ Reference 3)
  • Analysis of RNA by primer extension (see​ Reference 3)

Includes

  • RevertAid H Minus Reverse Transcriptase (200 U/μL) is supplied in storage buffer including: 50 mM Tris-HCl (pH 7.5), 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)

Note

Also available: RevertAid H Minus First Strand cDNA Synthesis Kit

  
Storage Condition-20 C
HazardousNo
Definition of Activity Unit
  • One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 minutes at 37°C.
  • Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/mL [3H]-dTTP, 0.4 mM polyA·oligo(dT)12-18.
InactivationInactivated by heating at 70°C for 10 minutes.
InhibitionMetal chelators, inorganic phosphate, pyrophosphate, and polyamines (see Reference 2)
Molecular Weight76.1 kDa monomer
Quality Control
  • The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
  • Functionally tested in first strand cDNA synthesis and RT-PCR.
SourceE. coli cells carrying a cloned fragment of the mutated pol gene encoding Moloney Murine Leukemia Virus reverse transcriptase.

References

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

  2. G. F. Gerard, J. M. D’Alessio, Methods in Molecular Biology (Humana Press, Totowa, NJ,1993), vol. 16, pp. 73-93.

  3. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, ed. 3, 2001). [third edition]

  4. A. Schmidt et al., UPS1 and UPS2 from Arabidopsis Mediate High Affinity Transport of Uracil and 5-Fluorouracil. J. Biol. Chem. 279, 44817-44824 (2004).

  5. K. G. Papavinasasundaram 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. R. Turk et al., Gene expression variation between mouse inbred strains. BMC Genomics. 5, 57 (2004).

  7. F. L. Pinto, P. Lindblad, A guide for in-house design of template-switch-based 5’rapid amplification of cDNA ends systems. Analytical Biochemistry. 397, 227-232 (2010).