FAQ: Why do some protocols recommend using CIP at 50°C and some recommend 37°C?

It has been shown that DNA fragments with 3´ extensions or blunt ends are slightly more difficult to dephosphorylate with CIP than those fragments with 5´ extensions. CIP works slightly better on 3´ extensions and blunt-ends at 50°C. We have found that the difference in efficiency is about 20-25%. Some protocols also recommend the addition of more CIP for these difficult ends. For the sake of simplicity, we recommend 37°C and an enzyme concentration of 0.5 unit/µg in a 10 µl reaction for all types of ends.