( ers ...)C. therm. Polymerase One-Step RT-PCR System

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• 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.

Experimental result 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. 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.
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