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Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler

Peter Scheinert, PhD, Barbara Behrens, PhD, and Dietmar Kahle, PhD
Peter Scheinert, PhD, Bernhard-Nocht-Institute for Tropical Medicine, Virology Dept., Bernhard-Nocht-Str. 74, D-20359 Hamburg
former address: University of Kiel, Institute for General Microbiology, Am Botanischen Garten, D-24118 Kiel
Barbara Behrens, PhD, and Dietmar Kahle, PhD, Eppendorf-Netheler-Hinz GmbH, Dept. Biotech Products, Barkhausenweg 1, D-22339 Hamburg

Abstract

The Polymerase Chain Reaction (PCR*) is a molecular-biological process that, during recent years, has been developed into a method used in virtually every area of medicine and natural sciences. This method was first described in 1985 (1) and enables selective in vitro amplification of a special DNA fragment, thereby emulating the cellular in vivo DNA replication. In spite of the method's basically simple operation, often enough it is not possible to achieve optimum results without optimizing the protocols. This summary therefore discusses a series of critical PCR parameters and feasible strategies for optimization.

Primer length and primer sequence

Prerequisite for amplification of a specific product is the selection of suitable primers. In this context, the following criteria are of essential importance:

The length of the primers;
the melting temperature (Tm);
the composition of the sequence and its physical properties; and
primer-primer interaction (2,3,4).
Ideal primers are between 18 and 30 bp long. The influence of the primer's melting temperatu
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