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One-Step Optimization Using Touchdown and Stepdown Polymerase Chain Reaction

  • Kenneth H. Roux
  • Karl H. Hecker
Protocol
  • 106 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Polymerase chain reaction (PCR) optimization and troubleshooting can consume considerable energy and resources because of the finicky and often unpredictable nature of the reactions. Small variations in any of the many variables in a given reaction can have a pronounced effect on the resultant amplicon profile. Reactions that are too stringent yield negligible product and reactions that are not stringent enough yield artifactual amplicons. Variables include concentrations of Mg2+, H+, dNTPs, primers, and template, as well as cycling parameters. With regard to the latter, the value selected for the annealing temperatures is most critical. Unfortunately, even with the most sophisticated algorithms (e.g., OLIGO) it is often difficult to predict the amplification optima a priori leaving no other choice but to employ empirical determination.

Keywords

Polymerase Chain Reaction Lower Annealing Temperature Conventional Polymerase Chain Reaction Polymerase Chain Reaction Protocol Annealing Stage 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Kenneth H. Roux
    • 1
  • Karl H. Hecker
    • 1
  1. 1.Department of Biological SciencesFlorida State UniversityTallahassee

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