Part of the
Methods in Molecular Biology
book series (MIMB, volume 193)
The Basics of RT-PCR
Some Practical Considerations
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.
KeywordsPhenol Electrophoresis MgCl2 Expense Cesium
Lo Y. M. D. (1998) Introduction to the polymerase chain reaction. Methods Mol. Biol.
, 3–10.Google Scholar
Lo Y. M. D. (1998) Setting up a PCR laboratory. Methods Mol. Biol.
, 11–17.Google Scholar
Chomczynski P., and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.
, 156–159.PubMedCrossRefGoogle Scholar
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
, 5294–5299.PubMedCrossRefGoogle Scholar
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
, 125–128.PubMedCrossRefGoogle Scholar