Product Class: Phosphatase

Alkaline Phosphatase, Calf Intestinal (CIP)

New! Quick Dephosphorylation Kit offers heat inactivation, high specific activity and a fast protocol.
icon_CutSmart incubation temp heat inactivation no
Catalog #SizeConcentration
M0290S1,000 units10,000 units/ml
M0290L5,000 units10,000 units/ml

Description

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.

Features

  • Faster reaction setup (no supplemental additives like zinc required)
  • Flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)
  • Less enzyme required (high specific activity), resulting in a lower cost per reaction
  • No need for multiple phosphatases (CIP removes 5´- and 3´- phosphates from DNA, RNA and dNTPs)
  • Active on unincorporated dNTPs in PCR products - improves DNA sequencing and 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

Applications

  • Dephosphorylation 5´ and 3´ ends of DNA and RNA.
  • 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.

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

-20°C

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

No

Molecular Weight

Theoretical: 69 kDa

Unit Assay Conditions

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

Heat Inactivated

No

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • 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.
  • 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

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Notes

  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.
  5. CIP activity is decreased in the presence of reducing agents (DTT, β-ME).

References

  1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed.). (p 5.72), Cold Spring Harbor Laboratory Press .
  2. Mossner, E., Boll, M. and Pfleiderer, G. (1980). Hoppe Seylers Z. Physiol Chem. 361
  1. Which alkaline phosphatase, Quick CIP, from the Quick Dephosphorylation Kit, 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, including CutSmart?
  4. Does the DNA need to be purified after the restriction digest, prior to CIP treatment?
  5. Can CIP be heat inactivated?
  6. Why do some protocols recommend using CIP at 50°C and some recommend 37°C?
  7. What phosphate groups are removed by rSAP, CIP, Quick CIP, or AnP?
  8. Does the DNA need to be purified after CIP treatment?
  9. Will Quick CIP, rSAP, CIP, or AnP dephosphorylate proteins?
  10. What is the effect of metal chelators, inorganic phosphate and phosphate analogs on CIP activity?”
  11. What is the effect of monovalent salts on CIP activity?
  12. What is the effect of reducing agents on CIP activity?
  1. Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290)