er/template system and cycler. Program parameters printed in bold should be kept invariable whereas other parameters are variable for primer/template-specific adaptation. Optimized values for variable program parameters on Eppendorf Mastercycler for a number of genomic and episomal targets are given in Table 4.

Table 3: Program parameters optimized for the 40 kb control PCR * Time increment of 20 seconds; for each elongation step the time is extended by 20 seconds (21 min 20 sec in 11th cycle; 21 min 40 sec in 12th cycle, etc).

Table 4: Variable cycling parameters optimized for various long targets 9.2 High fidelity PCR of targets between 100 bp 10 kb

For targets < 2 kb use the TripleMaster Polymerase Mix preferably in combination with the HighFidelity Buffer. For targets 2 kb < 10 kb test both the Tuning and HighFidelity Buffer and choose the combination that provides a better yield and/or specificity. If amplifying fragments < 500 bp using a low concentration of template DNA, increase the final buffer concentration in the PCR assay from 1x to 1.6x (8 l of 10x buffer in a 50 l reaction).

General hints

The fidelity of DNA amplification depends on the type of polymerase used and other factors, such as dNTP and Mg2+ concentration, template complexity and base composition, cycle number and pH of the reaction. The TripleMaster Polymerase Mix is optimized for high fidelity. Changing other reaction conditions, instead of changing the DNA polymerase, can further increase fidelity. Rules for optimiza
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