Alkaline Phosphatase   

Abstract

Materials and Reagents

1. Vector DNA
   
2. Restriction enzymes (New England Biolabs)
   
3. Alkaline phosphatase: Calf intestinal alkaline phosphatase (CIP) (New England Biolabs, catalog number: M0290 ) or Shrimp Alkaline Phosphatase (SAP) (Promega Corporation, catalog number: M8201 )
   

Procedure

1. Cut the vector (5 μg) in 100 μl digestion reaction.
   
2. Separate the digestion reaction to 2 tubes, 50 μl each.
   
3. Add 2.5 μl CIP enzyme or 1 μl SAP enzyme to 50 μl digestion (normally NEB digestion buffer is good for CIP enzyme, if not, purify the digestion and resuspend DNA in appropriate buffer).
   
4. Incubate at 37 °C for 1 h.
   
5. Heat inactivate the reaction at 65 °C for 30 min (for CIP) or 10 min (for SAP).
   
6. Gel purify the vector.
   Because CIP removes phosphate group, it is better to add some ATP during ligation step.