Phosphatase treatment of linearized vector

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[http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase]
[http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase]
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[[Category:Protocol]]
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[[Category:DNA]]
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[[Category:In vitro]]

Revision as of 14:52, 10 July 2006

To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with inserts.

Materials

Procedure

  1. Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
  2. Add 1μL Antarctic Phosphatase (probably should make final glycerol concentration less that 5%?)
  3. Incubate 60 mins at 37°C.
    This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.
  4. Heat-inactivate for 5 mins at 65°C.
  5. Proceed directly to ligation step.

Notes

NEB's Antarctic Phosphatase

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