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TripleMaster PCR System

g foils on PCR plates. Long-range PCR is sensitive to evaporation. 10.2 Little product on high background smear

- Reduce number of cycles by steps of 2. - Reduce template DNA by 50 ng decrements. - Increase the time of the elongation step (But do not increase the time extension per cycle in the long range PCR program). - Check the ratio of magnesium ions/dNTP. A final dNTP concentration at 500 M requires 2.5 mM Mg2+. - Reduce the amount of enzyme in 0.2 U decrements. - Increase the concentration of magnesium ions up to 3.5 mM in 0.25 increments if more than 500 ng template DNA, or more than 500 M dNTPs are used. 10.3 Low yield

- Choose the appropriate reaction buffer (see Table 1): Tuning Buffer for 240 kb PCR on genomic DNA and 1050 kb PCR on plasmid/phagemid DNA.
HighFidelity Buffer for 0.110 kb PCR on genomic DNA and plasmid/phagemid DNA. - Check the Tuning Buffer versus HighFidelity Buffer for GC-rich and other complex targets of any size between 210 kb. - Increase the final buffer concentration in the reaction assay to 1.6x (4 l of 10x HighFidelity or Tuning Buffer in 25 l or 8 l in 50 l, respectively) for targets < 5
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