( on. Start with 35 cycles if necessary...)TripleMaster PCR System

on. Start with 35 cycles, if necessary, increase up to 45 cycles.
² Determine the optimal annealing temperature empirically.
³Optimize elongation temperature. Increasing temperature >72C is not recommended for targets >5 kb due to temperature-driven damage of template DNA.
⁴ The elongation time depends on fragment length and template complexity. Use the upper values for each fragment size range given in Table 7 to amplify GC-rich targets. Further increase the elongation time if the product yield is still low or a background smear occurs.

10 Troubleshooting

Follow the manual as written!

10.1 No product

- Check the quality of primers and template. - Check the annealing and denaturation temperatures (especially for GC-rich targets). - Increase number of cycles (by steps of 5). - Increase template DNA by 50 ng increments. - Increase enzyme concentration (up to 2.5 U per 50 l). - Check the fit of the PCR tubes to the appropriate cycler. Bad contact affects temperature transfer. Use only consumables recommended by the cycler manufacturer. - Prevent air bubbles from becoming trapped after mixing the reaction Mastermixes. Air bubbles prevent homogenous temperature distribution throughout the reaction volume. - Check the fit of PCR tube caps or sealin
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