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HotStarTaq PCR Handbook

the yield of PCR product. 17. Problems with the thermal cycler Check the power to the thermal cycler and that the thermal cycler has been correctly programmed. Product is multi-banded 1. PCR cycling conditions not optimal Using the same cycling conditions, repeat the PCR using Q-Solution. 2. Annealing temperature too low Increase annealing temperature in 2C steps. Annealing time should be between 30 and 60 seconds. Difficulties in determining the optimal annealing temperature can be overcome in many cases by performing touchdown PCR (see appendix). 3. Primer concentration not optimal or
primers degraded Repeat the PCR with different primer concen trations from 0.10.5 M of each primer (in 0.1 M steps). In particular, when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel. 4. Primer design not optimal Review primer design Product is smeared
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