00 bp or when using a low amount of template DNA. - Increase the number of cycles (especially for complex genomic targets and GC-rich targets). - Increase the amount of template DNA per reaction by 50 ng increments. - Increase the amount of enzyme (up to 2.5 U per 50 l). - Increase the magnesium ions (up to 3.5 mM).
10.4 Non-specific product

- Try higher annealing temperatures. - Reduce the annealing time down to 8 seconds in 2 seconds decrements. - Reduce time of the elongation step (see Table 7). - Check Tuning Buffer versus HighFidelity Buffer (especially for targets < 2 kb). - Decrease the enzyme amount per reaction in 0.2 U decrements. - Reduce the amount of template DNA. - Reduce the number of cycles by steps of 2. - Add 12 % DMSO to a 50 l reaction. - Design new/longer primers.

11 Additional information

11.1 Reaction volume

The use of 0.2 ml thin-walled PCR tubes is recommended for long range PCR and other difficult PCR applications with a reaction volume of 50 l, and for high fideli
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