Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The Basic Polymerase Chain Reaction

(Protocol summary only for purposes of this preview site)

This protocol outlines the reagents and procedures required for a bog-standard PCR.

  • Primers. Each primer should be 2030 nucleotides in length and contain approximately equal numbers of the four bases, with a balanced distribution of G and C residues and a low propensity to form stable secondary structures. For further details, see Design of Oligonucleotide Primers for Basic PCR in the introduction to this chapter. As discussed below, restriction sites can be incorporated into the design of primers. Amplification using primers of this type generates DNAs that carry restriction sites at both ends, which can facilitate cloning.
    Oligonucleotide primers synthesized on an automated DNA synthesizer can generally be used in standard PCR without further purification. However, amplification of single-copy sequences from mammalian genomic templates is often more efficient if the oligonucleotide primers are purified by chromatography on commercially available resins (e.g., NENSORB; NEN Life Science Products) or by denaturing polyacrylamide gel electrophoresis, as described in Chapter 2, Protocol 3.
  • Template DNA. The template can be a fragment of DNA, a preparation of genomic DNA, a recombinant plasmid, or any other DNA-containing sample. Template DNA is dissolved in 10 mM Tris-Cl (pH 7.6) containing a low concentration of EDTA (<0.1 mM).
  • Thermostable DNA polymerase. Taq DNA polymerase is the standard and appropriate enzyme for the amplification stage of most forms of PCR. However, where elongation from 3-mismatched primers is suspected, a thermostable DNA polymerase with 35 proofreading activity may be preferred (Chiang et al. 1993). (See the section Thermostable DNA Polymerases above and the information panel Taq DNA Polymerase.)
    Taq DNA polymerase is supplied in a storage buffer containing 50 glycerol. Because this solution is very viscous and difficult to pipette accurately, the best method is to centrifuge the tube containing the enzyme at maximum speed for 10 sec at 4C in a microcentrifuge and then to withdraw the required amount of enzyme using a positive-displacement pipette.
  • Automatic pipetting devices. Automatic micropipetting devices equipped with barrier tips should be used to assemble the components of PCRs. Disposable barrier tips are fitted with a hydrophobic barrier to prevent the accidental passage of liquids into a micropipetting device. This arrangement reduces the potential for cross-contamination of PCR and DNA samples. Barrier micropipette tips are sold by several commercial companies (e.g., ART Barrier Tips from Molecular BioProducts).
  • Positive-displacement pipette. Positive-displacement pipetting, in which the piston is in direct contact with the liquid, is used for accurate transfer of high-viscosity liquids.
  • Microcentrifuge tubes or microtiter plates. Thin-walled plastic tubes that fit snugly in the block of the thermal cycler are used for amplification. Their use facilitates heat transfer and greatly reduces much of the time lag in reaching programmed temperatures.
  • Thermal cycler. A thermal cycler, programmed to carry out the desired amplification program, is required. A large number of programmable thermal cyclers marketed by different commercial companies are licensed by PerkinElmer for use in PCR. The choice among these instruments depends on the investigator's inclination, the available budget, and the range of uses to which the machine will be put. Before purchasing a thermal cycler, we recommend soliciting as many opinions as possible to discover the pros and cons of different machines.

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