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Team:Alberta/Notebook/protocols/vector dephos
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Latest revision as of 02:35, 27 October 2010
Vector Dephosphorylation
Reagents:
- Antarctic phosphatase
- 10x Antarctic phosphatase buffer
Procedure:
- Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer.
- Add 1ul of Antarctic phosphatase and mix.
- Incubate 5 minutes at 37oC.
- Heat inactivate for 5 minutes at 65oC.
- 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.
