A PCR Primer

PCR Enhancement Products Courtesy of RedasoftRedasoft's Visual Cloning 2000 includes primer design tools. Courtesy of Sigma-AldrichDirect loading of PCR products onto an agarose gel using Sigma-Aldrich's REDTaq. The art of PCR isn't hard to master. An abundance of products, ranging from relatively low-cost reagents to sophisticated optimization software, exists to meet most, if not all, PCR challenges. This profile looks at commonly used additives, PCR optimization kits and protocols, softwar

Written byAileen Constans

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The key to an optimal PCR experiment is primer design. In general, primers should be between 18 and 24 nucleotides long and have a GC content between 40 and 60 percent. Ideally primers should contain a relatively GC-rich 5'-end and an AT-rich 3'-end (i.e., containing three A or T residues in the last five bases).1 Designing primer pairs with complementary 3'-ends should be avoided, as this leads to the formation of primer dimers, which depletes the effective primer concentration. A number of companies have developed computer software to aid primer design. Some of these programs are available for free on the Internet. For example, both the Weizmann Institute of Science Bioinformatics Unit Web site (bioinformatics. weizmann.ac.il/mb/bioguide/pcr/software.html) and LabVelocity (www.labvelocity.com) provide links to various primer design programs. Integrated DNA Technologies (www.idtdna.com) offers a Web-based Oligo Analyzer that is built on technology from DNAStar Inc. of Madison, Wis., whereas Ulm, Germany-based Interactiva ...

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