( dNTP / Mg 2+ ...)TripleMaster PCR System

dNTP / Mg²⁺ Long range PCR: 500 M / 2.5 mM High fidelity PCR: 200 M / 2.5 mM (optional 80150 M/2.5 mM) PCR of GC-rich targets: 250500 M / 2.5 mM 11.5 Dilution of the TripleMaster Polymerase Mix

Prepare a 1:10 enzyme dilution as follows:
Add 32 l Molecular Biology Grade Water and 4 l of the corresponding 10x reaction buffer to a sterile microcentrifuge tube, mix well and spin down. Then add 4 l of the TripleMaster Polymerase Mix, mix thoroughly (glycerol solution) and spin down quickly. Keep the enzyme dilution on ice until use. It is stable for several hours. Do not store overnight!

11.6 Fidelity

In combination with both reaction buffers, the TripleMaster Polymerase Mix exhibits a 45 fold higher fidelity (2.3 x 10^-6) than Taq DNA polymerase (12.5 x 10^-6). The TripleMaster PCR System keeps the same high fidelity for long range amplification of large genomic targets (> 20 kb) as for amplification of short targets, because it circumvents the use of fidelityreducing co-solvents such as DMSO. Note: The addition of 5 % DMSO to the PCR causes a reduction of polymerase fidelity by more than 50 %. The additon of 10 % DMSO reduces the fidelity of proofreading polymerase blends to the level of Taq DNA polymerase alone.

11.7 Cloning

PCR products generated by the TripleMaster PCR System contain a mixture of fragments with blunt ends and 3-single nucleotide overhangs anal
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