Team:TU Darmstadt/Notebook/Methods/Dephosphorylation

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Revision as of 16:03, 17 October 2014

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Dephosphorylation

Short explanation:

Antarctic Phosphatase catalyzes the removal of 5' phosphate groups of DNA and RNA and thus prevents religation of cut vectors. It is used before ligation.

Procedure:

The reaction mix contains:

- up to 5 µg of digested DNA (no purification needed)

- 1/10 of reaction volume of 10x Antarctic Phosphatase Reaction Buffer

- 1 µL of Antarctic Phosphatase

- Incubate at 37° C for 30 minutes (3' ends) or 60 minutes (5' ends)

- Heat inactivate at 65° C for 15 minutes

- Continue with ligation

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