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When performing PCRs, usually in every protocol enzyme amount is specified as 1.25 U as optimal per 50 uL reaction. Then, when running 25 uL PCR reaction should i use 1/2 he amount the enzyme or should i respect the value on Units thus using the same volume as in 50 uL reaction?

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I usually use 1 unit per reaction in 20ul reactions. Using less makes the reaction less reliable in my experience. – Chris Mar 4 '14 at 16:50

You have to scale amounts of all PCR components proportionally depending on the volume of the reaction. The polymerase is no exception in this respect, the only difference is that you measure it in terms of activity - units. So in your protocol you have 0.025 U/µl and you have to maintain this concentration when changing the volume.

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