( ...)cMaster RTplusPCR System for increased sensitivity and product length ,,, in RT-PCR

Abstract

RT-PCR has become an essential method that allows researchers to determine the presence, structure and level of expression of an mRNA transcript in the organism, tissue or cell of interest. The basic protocol involves the synthesis of a cDNA copy of all the mRNA present in the sample, followed by amplification of the gene(s) of interest. Due to buffer incompatibilities, the two steps (RT and PCR) are often performed separately. While there are kits that combine the two steps into one, the limitations on sensitivity, due to the aforementioned buffer incompatibilities, are great. Eppendorf has designed a new kit with novel chemistries that allow for a more robust single-step RT-PCR reaction and even greater sensitivity in Two Step experiments. This paper examines the performance of this new kit.

Introduction

Reverse Transcription followed by PCR* (RT-PCR) is one of the most popular techniques for RNA analysis. One Step RT-PCR is the method of choice if many RNA samples need to be analyzed in parallel; the Two Step protocol is used for difficult targets or multiple target amplification from a single cDNA pool. The new Eppendorf cMaster RTplusPCR System enables the full range of RT-PCR applications. It contains a novel, recombinant reverse transcriptase, and a high fidelity PCR enzyme mix. In addition, the RTplusPCR buffer is formulated to adjust the Mg2+ concentration automatically, so that there is never a need for optimization of this critical component. This is achieved by weakly chelating Mg 2+ ions: when Mg2+ is present in excess, it is bound by the chelating agent, but as it is needed by the reaction (for Taq, or DNA), it is re
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