Phosphatase treatment of linearized vector
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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.
- Linear DNA from restriction digest (heat-inactivation of restriction enzymes is necessary but DNA purification is not).
- Antarctic Phosphatase
- 10X Antarctic Phosphatase buffer
- 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.