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Protocol/13
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Revision as of 00:42, 26 October 2010
Protocol 13: Vector dephosphorylation protocol
What you will need:
- Antarctic phosphatase
- 10x Antarctic phosphatase buffer
Procedure:
- Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5 ug of DNA cut with any restriction endonuclease in any buffer
- Add 1 ul of Antarctic phosphatase and mix
- Incubate 5 min at 37 C
- Heat inactivate for 5 minutes at 65 C
- Proceed with ligation
Notes
Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency. Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it. See also Sambrook.
