Phosphatase treatment of linearized vector-PDF

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 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