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
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.
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,
- 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).
- Sample: 50 pg to 5 g total RNA.
- Temperature optimum, reverse transcriptase reaction: 5072C.
- Divalent ion requirement: Mg2+.
- Works with UNG
(RT-PCR carry-over prevention).
- Accepts DIG-labeled and other modified nucleotides.
Comparison of C. therm Polymerase, Supplier
Ls RNase H - Reverse Transcriptase, and Supplier Ps 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 60C. Lane 2: Superscript II/Taq for 50 min
at 42C. Lane 3: AMV(Promega)/Taq for 60 min at 42C. Result: C. therm
works on templates where other systems fail.
- 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
- 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
Permits UNG-mediated carry-over prevention, because the system will
incorporate dUTP into amplified products that can then be removed by
in a subsequent PCR.
Source:Page: All 1 2 3 Related biology technology :1
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A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning6
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. High-Fidelity PCR with a Novel Polymerase Mixture9
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. The Use of MasterTaq Polymerase for PCR with Humic Material-Contaminated