Product Class: Other

Antarctic Phosphatase
NEBU cloned at NEB recombinant 37 80 Heat

Product Introduction

Antarctic Phosphatase (AnP) is a heat labile alkaline phosphatase purified from a recombinant source.

  • Rapid and irreversible heat inactivation eliminates unwanted activity
  • Flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)
  • No need for multiple phosphatases (AnP removes 5´- and 3´- phosphates from DNA, RNA and dNTPs)
  • Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysis

Catalog # Size Concentration
M0289S 1000.0 units 5000 units/ml
M0289L 5000.0 units 5000 units/ml

Product Information

Description

Antarctic Phosphatase (AnP) is a heat labile alkaline phosphatase purified from a recombinant source. AnP nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. Also, AnP Hydrolyses ribo-, as well as deoxyribonucleoside triphosphates (NTPs and dNTPs). AnP 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. AnP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis. AnP is completely and irreversibly inactivated by heating at 80°C for 2 minutes, thereby making removal of AnP prior to ligation or end-labeling unnecessary. 

Product Source

An E. coli strain that carries the TAB5 AP gene, originally cloned in plasmid pNI (2), recloned in plasmid pEGTAB7-4.1(3).
This product is related to the following categories:
Phosphatases Products,
RNA Modification,
This product can be used in the following applications:
Dephosphorylation

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme that will dephosphorylate 1 µg of pUC19 vector DNA cut with a restriction enzyme generating 5´ recessed ends in 30 minutes at 37°C. Dephosphorylation is defined as > 95% inhibition of recircularization in a self-ligation reaction and is measured by transformation into E. coli.

Reaction Conditions

1X Antarctic Phosphatase Reaction Buffer
Incubate at 37°C

1X Antarctic Phosphatase Reaction Buffer
50 mM Bis-Tris-Propane-HCl
1 mM MgCl2
0.1 mM ZnCl2
(pH 6 @ 25°C)

Storage Buffer

10 mM Tris-HCl
1 mM MgCl2
50% Glycerol
0.01 mM ZnCl2
pH 7.4 @ 25°C

Heat Inactivation

80°C for 2 minutes

Molecular Weights

Apparent: 35 kDa
Theoretical: 69 kDa

Unit Assay Conditions

Vector DNA is dephosphorylated in restriction endonuclease buffer supplemented with Antarctic Phosphatase Reaction Buffer. Ligation is performed with 50 ng of vector using the NEB Quick Ligation Kit (NEB #M2200).

Application Features

  • 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.

Product Notes

  1. Adding 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer will provide the amount of Zn2+ that the enzyme requires for activity.
  2. Antarctic Phosphatase is also active in all restriction enzyme NEBuffers 1.1, 2.1, 3.1 and CutSmart® Buffer only when supplemented with 1/10 volume of the 10X Antarctic Phosphatase Reaction Buffer.
  3. Antarctic Phosphatase activity is enhanced in the presence of monovalent salts.
  4. Antarctic Phosphatase is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs. Antarctic Phosphatase activity is decreased in the presence of reducing agents (DTT, β-ME).
  5. Molecular Weight: Antarctic Phosphatase is a homodimer. The molecular weight of the monomer is 35 kDa.
  6. Antarctic Phosphatase, as are most alkaline phosphatases, is a Zn2+ and Mg2+ –dependent enzyme and does require supplemental zinc.

References

  1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual,
    . (2nd ed.), 5.72.
  2. Rina, M. et al. (2000). Eur. J. Biochem.. 267, 1230-1238.
  3. Guthrie, E., unpublished results. Unpublished observation

Protocols, Manuals & Usage

Protocols

  1. Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Which alkaline phosphatase, Quick CIP, rSAP or Antarctic Phosphatase works best?
  2. The number of colonies that don't contain an insert seems high, how can I tell if the Antarctic Phosphatase worked?
  3. Will Quick CIP, rSAP or Antarctic Phosphatase (AnP) dephosphorylate proteins?
  4. Does the DNA need to be purified after the restriction digest, prior to Antarctic Phosphatase treatment?
  5. Does the DNA need to be purified after Antarctic Phosphatase treatment?
  6. What is the molecular weight?
  7. Can Antarctic Phosphatase be heat inactivated?
  8. What phosphate groups are removed by rSAP, Quick CIP, or Antarctic Phosphatase (AnP)?
  9. Does the DNA need to be purified after a restriction digest and prior to the dephosphorylation step?
  10. Does the DNA need to be purified after the dephosphorylation step and prior to the ligation step?
  11. Are the alkaline phosphatases active in NEBuffers?
  12. Cloning Problem:  Too much background in the transformation step.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

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]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.