( 1 some app...)TripleMaster PCR System

¹ some applications may require an extra addition of Magnesium Solution (supplied) to set up a final concentration >2.5 mM.
² increase final buffer conc. (1.6x) for targets < 500 bp or when using a low concentration of template DNA per
reaction (< 10 ng genomic DNA or < 0.5 ng plasmid DNA)

Cycling program for high fidelity PCR

A standard program which can be adapted to an appropriate primer/template system is shown in Table 6. The data of Table 7 may be used as a guide for the adaptation of individual PCR reactions.

Table 6: Program for high fidelity PCR with Eppendorf Mastercycler ¹ Number of cycles depends on product yield required, template concentration and complexity.
² The annealing temperature depends on the melting temperatures of the primer set used.
³ This elongation temperature is recommended for long targets >5 kb to reduce the temperature-driven damage of template DNA.
⁴ The elongation time depends on fragment length and type of thermal cycler. Elongation times with the Eppendorf astercycler for various fragment sizes are given in Table 7. These values can also be transferred to other Peltier-driven cyclers.

Table 7: Target size and elongation time ¹ The elongation time required for targets with an unusual (non-random) nucleotide base composition, repeats or high GC-content is longer than for standard targets of the same size. In those cases, work at the upper limit of time range given.


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