( tion of general reaction conditions to ...)TripleMaster PCR System

tion of general reaction conditions to achieve maximum fidelity are provided below:

- Start with 200 M of dNTP concentration. For smaller targets up to 2 kb try final concentrations of 80, 100, 120 and 150 M to further increase the PCR fidelity. - Run only as many cycles as necessary for obtaining enough product: 30 cycles for single-copy genomic targets, 25-28 cycles for plasmid/phage targets or multicopy genomic targets. - Increase initial template concentration to obtain more product. - Use high quality dNTPs only. dNTP solutions which are contaminated by chemically altered bases, pyrophosphate, salts, rNTPs, dNDPs and dNMPs lead to misincorporation, mispairing, strand termination, nucleotide modification and excision and inhibit the DNA polymerases. dNTP contamination has a direct impact on DNA amplification fidelity. - Avoid excess of enzyme in amplification reactions. For high fidelity PCR use 0.51.0 U per 50 l (see Table 5). Empirically determine the lowest possible amount of enzyme to obtain enough product and sensitivity for an individual application. - Variable reaction volumes (20, 25, 50, or 100 l) and tube formats (0.2 or 0.5 ml) can be used. Adjust the amount of enzyme per reaction respectively (see Table 5). Note: Adjusting reaction conditions for maximal PCR fidelity always leads to a reduction of product yield!

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