Phosphatase treatment of linearized vector: Difference between revisions
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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] | ||
[[Category:Protocol]] | |||
[[Category:DNA]] | |||
[[Category:In vitro]] |
Revision as of 11:52, 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
- Linear DNA from restriction digest (heat-inactivation of restriction enzymes is necessary but DNA purification is not).
- Antarctic Phosphatase
- 10X Antarctic Phosphatase buffer
Procedure
- Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
- Add 1μL Antarctic Phosphatase (probably should make final glycerol concentration less that 5%?)
- 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. - Heat-inactivate for 5 mins at 65°C.
- Proceed directly to ligation step.