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For a complete description of primers, PCR programs and a discussion of the PCR conditions please consult: Andrologia 26: 97-106 (1994) and Biotechniques 23: 504-511 (1997). Click here to get the Biotechniques paper in PDF format.

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 PCR buffers

A commonly used PCR buffer, includes only KCl, Tris and MgCl2 (for example, Perkin Elmer Cetus); a somewhat more complex buffer was previously proposed for multiplex reactions of the DMD gene exons (Chamberlain et al. (1988) in Nucleic Ac Res 16: 11141-11156). These buffers were compared in multiplex PCR reactions, for their efficiency in supporting the activity of the Taq polymerase. Figure 15 shows that, PCR reactions on four different genomic DNA templates were consistently more efficient (more PCR product) when performed in 1.6x PCR buffer than 1x DMD buffer. Same amount of template DNA and primer were taken in all reactions, which were run in the same conditions at the same time. The same amount of product was loaded in each lane on the gel.

Table 3. Comparison of PCR buffers.
10x PCR buffer
5x DMD buffer
  • 500 mM KCl
  • 100 mM Tris-HCl (pH 8.3)
  • 15 mM MgCl2
  • 83mM (NH4)2SO4
  • 335mM Tris-HCl (pH8.8)
  • 33.5mM MgCl2
  • 50mM ß-Mercapthoethanol
  • 34 mM EDTA
  • optimal dNTP concentration in the reaction = 200 mM
  • optimal dNTP concentration in the reaction = 6000 mM

Fig 15. Comparison of two differnt PCR buffers. Multiplex mixture F was used in PCR amplification of 4 different genomic DNA templates. Reactions were performed in identical conditions with the exception of the buffers. Results indicate a higher yield of products in reactions performed in 1.6 x PCR buffer.