Phosphatase Removal of PO4 groups from DNA

Contributed by Ivan Delgado <>

The removal of phosphate groups from DNA using Alkaline Phosphatase

1 unit of Alkaline Phosphatase (AP) can hydrolyze 50 pmol of 5’ terminal phosphorylated DNA fragments (3’ recessed, 5’ recessed or blunt-ended) when incubated at 37oC for 1 hour. This implies that 25 pmol of DNA are dephosphorylated (at both ends) in one hour by 1 unit of AP at 37oC


10X Dephosphorylation buffer (0.5 M Tris-HCl, pH 8.5, 1 mM EDTA) Alkaline phosphatase


  • This protocol takes into account the prior preparation of a DNA fragment, such as one isolated from low melting agarose
  • Phosphatase treat appropriate samples (for example: if digested vector can religate, i.e. digested by a single enzyme): mix 20 mL of 10X dephosphorylation buffer, 10 uL of Calf alkaline phosphatase (1U/uL) (contains 50% glycerol, so the volume of AP used should never be more than 10% of the final volume), and ~170 uL of Vector + dH2O (fragment) (assuming ~0.5 ug) (200 uL total volume)
  • Incubate at 37oC for 1 hour
  • Add 0.1X Vol of 200mM EDTA/EGTA (in this case, 20 uL) and HEAT inactivate at 65oC for >10 mins. The DNA is ready for downstream applications

NOTE: the EDTA/EGTA should not interfere with ligation reactions if only 0.1X Vol of the AP reaction is used in the ligation reaction (no purification step in between, see DNA Ligation), in which case the EDTA/EGTA would have been dropped down to 1 to 2mM)NOTE: Note: when determining the amount of DNA to use: 1) determine concentration by spectrophotometer or better 2) run an aliquots (1, 5, 10 uL) on a gel. Determine the amount that is barely visible (EtBr has a visibility limit of about 100-200 ng on agarose gels) on a THIN gel (thick gels will mask some of the signal, specially at low DNA concentrations). Use two to three times more for the AP reaction. For example: if the aliquot that could be detected in the gel was 5 uL, then use 10 to 15 uL and dilute with dH2O to the appropiate volume (in the example above, to 170 uL with 160 to 155 uL dH2O)

This method is based, with permission, on an original protocol available here.