From 2010.igem.org
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| - | Protocol 13: Vector Dephosphorylation
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| - | Reagents:
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| - | * Antarctic phosphatase
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| - | * 10x Antarctic phosphatase buffer
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| - | Procedure:
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| - | * Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer.
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| - | * Add 1ul of Antarctic phosphatase and mix.
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| - | * Incubate 5 minutes at 37<sup>o</sup>C.
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| - | * Heat inactivate for 5 minutes at 65<sup>o</sup>C.
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| - | * Proceed with ligation.
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| - | Notes:
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| - | * Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh.
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| - | * It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency.
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| - | * Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it.
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| - | ** See also Sambrook.
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| - | [[Team:Alberta/Notebook/protocols| Back]]
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| - | {{Team:Alberta/endMainContent}}
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Latest revision as of 17:13, 26 October 2010
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