Dephosphorylation prior to cloning

In cloning protocols, a DNA fragment is ligated into a plasmid vector. Especially if the vector is cut with a single restriction enzyme, chances are much higher that the vector religates back on itself rather than on an added DNA fragment. This results in a high fraction of “empty clones”, or background.

Alkaline phosphatases are routinely used to reduce the background from empty, religated vectors during cloning of DNA fragments, since dephosphorylated DNA termini cannot be ligated by DNA ligase. The phosphatase treatment will effectively reduce the background of “empty” clones by >95%. However, cloning procedures and also phosphatase treatment may be cumbersome and error prone.

SAP offers greater convenience to this procedure, since the enzyme may be completely removed by a simple heat inactivation step. SAP is active in all buffers used for restriction enzymes, so SAP can be added either during restriction digestion, or directly after. With SAP, the user can forget elaborate calculations and multi-step incubations, because the enzyme completely dephosphorylates DNA during one, simple, incubation.

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There are many ways to use SAP for vector dephosphorylation, and we recommend two protocols below. These protocols apply to all types of DNA termini; 3’-protruding, blunt or 5’-protruding.

Protocol including restriction cutting

In this protocol, SAP is present during restriction cutting, so the termini are dephosphorylated as soon as they are formed. In this protocol, the minimum effective amount of SAP is proportional to the amount of restriction enzyme added (i.e. the rate of terminus formation). In the simple sense, use at least 0.1 U SAP per Unit restriction enzyme, and proceed to complete cutting. The amount of restriction enzyme may differ from the protocol below, please use amounts recommended by the supplier.

  • 1 µg plasmid
  • 5 Unit restriction enzyme
  • 5 µl 10x restriction enzyme buffer
  • 1 Unit SAP
  • dH2O to 50 µl

Incubate at 37°C for 1 hour, inactivate as recommended for the restriction enzyme used. Proceed to ligation protocol.

Quick dephosphorylation of cut plasmid

Efficient dephosphorylation can be achieved in short time using high amounts of SAP. When restriction cutting is complete, simply add 5 U SAP per µg vector to your restriction mix and incubate for further 5 min at 37°C. Inactivate as recommended for the restriction enzyme. Proceed to ligation protocol.