( for one-step ...)Titan One Tube RT-PCR System / Kit

for one-step RT-PCR amplification of up to 6 kb targets Cat. No. 1 888 382 25 reactions

Cat. No. 1 855 476100 reactions
Cat. No. 1 939 823 50 reactions Description Components of the Titan One Tube RT-PCR System/Kit allow completion of RT-PCR in a single tube, one-step reaction. The system includes the following components:

• Enzyme mix containing AMV Reverse Transcriptase (for reverse transcription) and the Expand High Fidelity PCR System (Taq/Pwo enzyme blend, for PCR)
• Additional reaction components (RT-PCR reaction buffer with 7.5 mM MgCl₂; MgCl₂ solution; dithiothreitol solution) for convenient, quick reaction set-up

In addition to all the components of the Titan One Tube RT-PCR System, the Titan One Tube RT-PCR Kit also contains:

• PCR Grade dNTP mix
• PCR grade water
• RNase Inhibitor; human control RNA
• Control primer mix for amplification of a human beta-actin product

Application The Titan One Tube RT-PCR System or Kit can generate up to 6 kb PCR products from total RNA, mRNA, or viral RNA. The system may be used for:

• Amplification of specific RNA targets
• Cloning of RNA-sequences without constructing a cDNA library
• Competitive or multiplex PCR.

Application profile Operating parameters

• Starting samples: 1 pg to 1 g total RNA
• Temperature optimum, reverse transcriptase reaction: 50C
  Note: The enzyme may be used at temperatures up to 60C
• Requires two sequence-specific primers
• Accepts DIG-labeled and other modified nucleotides

Note: The Titan One Tube RT-PCR System/Kit is not recommended for carry-over prevention using dUTP and Uracil-DNA Glycosylase Experimental result RT-PCR of beta-actin transcript from K562 cells. mRNA from serially diluted cell lysates was captured in streptavidin-coated PCR tubes using the mRNA Capture Kit. RT-PCR was performed with the Titan System (RT at 50C) or RNase H^- M-MuLV reverse transcriptase from Supplier L (RT at 42C) +Taq DNA polymerase according to each suppliers protocol. The Titan System delivers > 100 times greater sensitivity than the two step method with Supplier L. Key advantages

• Significantly reduces time needed to perform RT-PCR, because the one-step reaction minimizes handling and reduces the risk of contamination
• Improves the specificity and sensitivity of cDNA synthesis, because the elevated temperature of the AMV Reverse Transcriptase reaction eliminates secondary structure in the template and increases primer specificity
• Increases yield and fidelity of PCR amplification, because the Taq/Pwo enzyme blend gives threefold greater fidelity and twofold higher yields than Taq DNA Polymerase alone

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