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Team:TU Darmstadt/Notebook/Methods/Dephosphorylation
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Latest revision as of 00:29, 18 October 2014
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
