When should I add the polymerase for hot start PCR?

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5 years ago

Maryam Amir

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When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?

Experiment: PCR Hot start PCR - Bacterial DNA

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**Stefan Fuhrmann** · Answer 1 · 2019-11-07T20:38:00+00:00

For a hot start PCR you need to prepare the mix without the enzyme. Then at the start of the first annealing step, you should add your polymerase. In this way, your polymerase will not affect any material that annealed on temperature below the one you intended.