The Polymerase Chain Reaction

Martha F. Kramer1, Donald M. Coen1

1 Harvard Medical School, Boston
Publication Name:  Current Protocols in Protein Science
Unit Number:  Appendix 4J
DOI:  10.1002/0471140864.psa04js29
Online Posting Date:  November, 2002
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Abstract

This appendix describes the methodology behind PCR and gives a method for amplifying DNA enzymatically by the PCR and for optimizing this reaction for the sequence and primer set of interest.

     
 
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Table of Contents

  • Basic Protocol 1: Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
  • Reagents and Solutions
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

  Materials
  • 10× MgCl 2‐free PCR buffer (see recipe)
  • 50 µM oligonucleotide primer 1: 50 pmol/µl in sterile H 2O (store at −20°C)
  • 50 µM oligonucleotide primer 2: 50 pmol/µl in sterile H 2O (store at −20°C)
  • Template DNA: 1 µg mammalian genomic DNA or 1.0 to 100.0 pg of plasmid DNA
  • recipe25 mM 4dNTP mix (see recipe)
  • 5 U/µl Taq DNA polymerase (native or recombinant)
  • recipeEnhancer agents (optional; see recipe)
  • 15 mM (L), 30 mM (M), and 45 mM (H) MgCl 2
  • Mineral oil
  • TaqStart Antibody (Clontech)
  • Ficoll 400 (optional): prepare as 10× stock; store indefinitely at room temperature
  • Tartrazine dye (optional): prepare as 10× stock; store indefinitely at room temperature
  • 0.5 ml thin‐walled PCR tubes
  • Automated thermal cycler
NOTE: Do not use DEPC to treat water, reagents, or glassware.NOTE: Reagents should be prepared in sterile, disposable labware, taken directly from its packa ging, or in glassware that has been soaked in 10% bleach, thoroughly rinsed in tap water followed by distilled water, and if available, exposed to UV irradiation for ∼10 min. Multiple small volumes of each reagent should be stored in screw‐cap tubes. This will then serve as the user's own optimization “kit.” Thin‐walled PCR tubes are recommended.
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Figures

Videos

Literature Cited

Literature Cited
   Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1988. Primer‐directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487‐491.
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