ty PCR applications of 2025 l.

11.2 Use of PCR additives and co-solvents with TripleMaster PCR System

In most cases, the TripleMaster PCR System produces good results without any additive, with either the Tuning or HighFidelity Buffer. For very GC-rich (>70%) templates or templates containing complex secondary structure, the addition of iso-stabilizing cosolvents such as betaine (0.53 M final concentration), DMSO (25 % final), glycerol (25%), trimethylamine N-oxide (10100 M) or trimethyleamine hydrochloride (10100 M) may improve yield and specificity of PCR. However, the use of destabilizing co-solvents substantially reduces the PCR fidelity and requires a re-optimization of the annealing temperature. The TaqMaster PCR Enhancer, available separately, can be used to stabilize the enzyme for elevated denaturation temperatures (up to 98C) during amplification of GCrich targets. This approach is not feasible in long range PCR.

11.3 Enzyme concentration

Long range applications: 2 U = 0.4 l Other applications: 0.52.5 U = 0.10.5 l 11.4 Concentrations of dNTPs and Mg²⁺

Both buffers of the TripleMaster PCR System provide a final Mg concentration of 2.5 mM which is ideal for most applications. An addition of 25 mM Magnesium Solution to the PCR reactions (in increments of 0.2 mM) is necessary only if the template concentration exceeds 500 ng per 50 l PCR volume and/or the dNTP concentration is higher than 500 M.


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