Team:Alberta/Notebook/protocols/vector dephos

From 2010.igem.org

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* It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency.  
* 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.  
* 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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* See also Sambrook.
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Revision as of 00:44, 27 October 2010

TEAM ALBERTA

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.


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