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The Basics of RT-PCR

Some Practical Considerations
  • Joe O’Connell
Part of the Methods in Molecular Biology book series (MIMB, volume 193)

Abstract

The basic reverse-transcriptase-polymerase chain reaction (RT-PCR) technique is used routinely and widely in most biomedical research laboratories. The fundamental considerations for such basics as primer design, good laboratory set up and practice, and techniques for performing RT-PCR have been fully examined elsewhere (1,2). Thus, I will include only observations from my own experience in this chapter. Although the purpose of this chapter is to help the reader in setting up their own PCR assays, and to provide tips on effective PCR laboratory set up, it should be noted that each individual chapter in this volume contains complete experimental detail on the protocols described.

Keywords

Optimal Annealing Temperature Guanidine Thiocyanate Household Bleach Nonspecific Priming Cesium Chloride Density Gradient 
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.

References

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    Lo Y. M. D. (1998) Introduction to the polymerase chain reaction. Methods Mol. Biol. 16, 3–10.Google Scholar
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    Lo Y. M. D. (1998) Setting up a PCR laboratory. Methods Mol. Biol. 16, 11–17.Google Scholar
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    Chomczynski P., and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.PubMedCrossRefGoogle Scholar
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    Chirgwin J. M., Przybyla A. E., MacDonald R. J., and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294–5299.PubMedCrossRefGoogle Scholar
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    Longo M. C., Berninger M. S., and Hartley J. L. (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125–128.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Joe O’Connell
    • 1
  1. 1.Department of MedicineNational University of IrelandCorkIreland

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