Product Class: Phosphatase

Alkaline Phosphatase, Calf Intestinal (CIP)

incubation temp heat inactivation no
Catalog #SizeConcentration
M0290S1,000 units10,000 units/ml
M0290L5,000 units10,000 units/ml


Alkaline Phosphatase, Calf Intestinal (CIP) nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. Also, CIP hydrolyses ribo-, as well as deoxyribonucleoside triphosphates (NTPs and dNTPs). CIP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA. The enzyme acts on 5´ protruding, 5´ recessed and blunt ends. CIP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.

Product Source

Calf intestinal mucosa

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X

Advantages and Features


  • Dephosphorylation of cloning vector DNA to prevent recircularization during ligation
  • Dephosphorylation of DNA prior to end-labeling using T4 Polynucleotide Kinase
  • Treatment of dNTPs in PCR reactions prior to sequencing or SNP analysis
  • Dephosphorylation of DNA and RNA

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme that hydrolyzes 1 μmol of p-Nitrophenyl Phosphate, PNPP (NEB #P0757) in a total reaction volume of 1 ml in 1 minute at 37°C.

Reaction Conditions

1X CutSmart® Buffer
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Storage Temperature


Storage Conditions

10 mM Tris-HCl
50 mM KCl
1 mM MgCl2
50% Glycerol
0.1 mM ZnCl2
pH 8.2 @ 25°C

Heat Inactivation


Molecular Weight

Theoretical: 69 kDa

Specific Activity

3,000 units/mg

Unit Assay Conditions

1 M diethanolamine- HCl (pH 9.8), 0.5 mM MgCl2, 50 mM p-nitrophenylphosphate and enzyme. These conditions are only used for quantitating enzyme activity.

Heat Inactivated


Quality Control

Quality Control Assays

  • Endonuclease Activity (Nicking):
  • Exonuclease Activity (Radioactivity Release):
  • Functional Test (Dephosphorylation):
    The Alkaline Phosphatase is tested in a reaction with linearized DNA.  After incubation the DNA is ligated and transformed into competent cells.  The degree of dephosphorylation is determined by comparison to linearized DNA that is not treated with the alkaline phosphatase.
  • Protein Purity (SDS-PAGE):
  • RNase Activity (2 Hour Digestion):
    The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

Supporting Documents



  1. CIP, as are most alkaline phosphatases, is a Zn2+ and Mg2+-dependent enzyme. Our formulation of its storage buffer provides Zn2+ and Mg2+, which does not require supplemental zinc or other additives in reactions with CIP.

  2. CIP is also active in 1X NEBuffers 1.1, 2.1, 3.1, as well as NEBuffers 1, 2, 3, 4 and unique NEBuffer for EcoRI.

  3. CIP activity is enhanced in the presence of monovalent salts.

  4. CIP is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs.


  1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed,). (p5.72), Cold Spring Harbor Laboratory Press .
  2. Mossner, E., Boll, M. and Pfleiderer, G. (1980). Hoppe Seylers Z. Physiol. Chem.. 361
  1. Which alkaline phosphatase, rSAP, CIP, or Antarctic Phosphatase, works best?
  2. The number of colonies that do not contain an insert seems high, how can I tell if the CIP worked?
  3. Will CIP work in restriction enzyme NEBuffers?
  4. Does the DNA need to be purified after the restriction digest, prior to CIP treatment?
  5. Does the DNA need to be purified after CIP treatment?
  6. Will CIP dephosphorylate proteins?
  7. Can Antarctic Phosphatase remove the 3' phosphate from DNA?
  8. Can CIP be heat inactivated?
  9. Why do some protocols recommend using CIP at 50°C and some recommend 37°C?
  1. Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction (M0290)