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C. Therm. Polymerase One-Step RT-PCR System

C. therm. Polymerase One-Step RT-PCR System
for one-step RT-PCR amplification of up to 3 kb RNA targets Cat. No. 2 016 338      50 reactions
Cat. No. 2 016 346    250 reactions
 
Description
The system contains a unique enzyme blend of C. therm. Polymerase and Taq DNA Polymerase.

Note: 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. The system also contains RT-PCR reaction buffer (5x conc.) with 12.5 mM MgCl2 and 10% DMSO; DMSO solution; and 100 mM DTT solution.
 
Application
The C. therm. Polymerase One-Step RT-PCR System may be used for a one-tube, one-step RT-PCR. The C. hydrogenoformans DNA polymerase produces first strand cDNA with high yield and specificity, then Taq DNA Polymerase amplifies the cDNA. The system may be used for:
  • Amplification of up to 3 kb targets (especially GC-rich sequences) from mRNA or total RNA for virus detection, cloning, mutation analysis, etc.
  • Qualitative, semi-quantitative, or quantitative analysis of RNA transcription levels (in combination with gel-based or ELISA detection methods).
  • RT-PCR in the presence of dUTP for prevention of carry-over contamination in combination with UNG (see the pack insert for details).
Application profile
 
 
Operating parameters
  • Sample: 50 pg to 5 µg total RNA.
  • Temperature optimum, reverse transcriptase reaction: 50°–72°C.
  • Divalent ion requirement: Mg2+.
  • Works with UNG (RT-PCR carry-over prevention).
  • Accepts DIG-labeled and other modified nucleotides.
Experimental result
 
 
Comparison of C. therm Polymerase, Supplier L’s RNase H - Reverse Transcriptase, and Supplier P’s AMV Reverse Transcriptase for RT-PCR. Template: Total RNA isolated from K562 cell line. Primer: TGF-beta. Target: 650 bp product. 1.5 M betaine to dissolve secondary structure. Positive Controls: 1 ng of a 997 bp beta-actin fragment from mouse. Lane 1: C. therm. polymerase/Taq for 30 min at 60°C. Lane 2: Superscript II/Taq for 50 min at 42°C. Lane 3: AMV(Promega)/Taq for 60 min at 42°C.  Result: C. therm works on templates where other systems fail.
 
Key advantages
  • Extends the sensitivity of RT-PCR, because the system can detect as few as 100 copies of a template (e.g. viral RNA).
  • Ensures high specificity amplification, because the system will accurately amplify target RNA even in the presence of large amounts of non-specific RNA.
  • Efficiently transcribes GC-rich RNA regions, due to the high temperature of the cDNA reaction and the presence of DMSO in the reaction buffer.
  • Ensures high fidelity RT-PCR, because the C. therm. Polymerase One-Step RT-PCR System has a twofold lower transcriptional error rate than Tth DNA Polymerase.
  • Permits UNG-mediated carry-over prevention, because the system will incorporate dUTP into amplified products that can then be removed by UNG in a subsequent PCR.

 

Roche Contact Information

Roche
Roche Diagnostics Corporation
P.O. Box 50414
9115 Hague Road
Indianapolis, IN 46250-0414 USA




Customer Service: 800-262-1640

Tech Support: 800-262-4911

Fax Number: 800-428-2883

Web Site: http://www.roche-applied-science.com
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