Phosphatase treatment of linearized vector

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

 * 1) Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
 * 2) Add 1&mu;L Antarctic Phosphatase (probably should make final glycerol concentration less that 5%?)
 * 3) Incubate 60 mins at 37&deg;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&deg;C.
 * 5) Proceed directly to ligation step.