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PCR reaction components

.html">PCR Nucleotide Mix, Cat. No. 1581295) may be added to the reaction mixture as a single reagent. In addition, individual PCR Grade dNTPs and a set of PCR Grade dNTPs (Cat. No. 1969064) are available. If you increase the concentration of dNTPs you must also increase Mg2+ concentration. Increases in dNTP concentration reduce free Mg2+, thus interfering with polymerase activity and decreasing primer annealing. For prevention of carry-over contamination a higher concentration of dUTP is usually used in place of dTTP (for details, see Preventing carry-over contamination with uracil-DNA glycosylase). The final dNTP concentration should be 50500 M (each dNTP). The most commonly used dNTP concentration is 200 M. pH Generally, the pH of the reaction buffer supplied with the corresponding thermostable DNA polymerase (pH 8.39.0) will give optimal results. However, for some systems, raising the pH may stabilize the template and enhance results. Note: Determining the optimal reaction pH can be simplified with the PCR Optimization Kit from Roche Applied Science (Cat. No. 1636138). Reaction additives In some cases, adding the following compounds can enhance the efficiency or specificity of PCR:
  • Betaine (0.52 M)
  • Bovine serum albumin (BSA; 100 ng/50 l)
  • Detergents
  • Dimethylsulfoxide (DMSO; 210%) (v/v)
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