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C. therm. Polymerase for Reverse Transcription in Two-Step RT-PCR

Polymerase for two-step RT-PCR amplification of up to 4 kb targets

Cat. No. 2 016 311 300 units

Description C. therm. Polymerase provided by Roche Applied Science is the recombinant form of the DNA polymerase from the thermophilic eubacterium Carboxydothermus hydrogenoformans, expressed in E. coli. The recombinant enzyme lacks the 5'3' exonuclease domain (RNase H activity) of the native enzyme. Application Produces first strand cDNA (for RT-PCR) or full-length double-stranded cDNA transcripts from total RNA or mRNA. May be used for:
  • Two-step RT-PCR for virus detection, cloning, mutation analysis, etc.
  • One-step RT-PCR (e.g. when used with Taq DNA Polymerase).
  • One-Step RT-PCR System.
  • RT-PCR amplification of GC-rich sequences.
Operating parameters
  • Temperature optimum, reverse transcriptase reaction: 5572C.
  • Divalent ion requirement: Mg2+.
  • Addition of betaine (0.52.0 M) to reaction mix improves yield and specificity of transcription for GC-rich templates.
  • Accepts DIG-labeled and other modified nucleotides.
  • Works with Uracil-DNA Glycosylase (PCR carry-over prevention).
Key advantages
  • Enhances specificity, because the high reaction temperature reduces nonspecific primer binding.
  • Ensures high fidelity first strand cDNA, because the enzyme has a twofold lower transcriptional error rate than Tth DNA Polymerase.
  • Efficiently transcribes GC-rich mRN A regions, because the thermoactive enzyme can operate at temperatures (up to 72C) high enough to eliminate most secondary structure, even in GC-rich regions.
  • Accurately transcribes even low amounts of template (high sensitivity).
  • Flexible, because the enzyme can use either oligo(dT) primer or anchor primer at temperatures up to 60C.



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