Quick Dephosphorylation Kit

Description

The Quick Dephosphorylation Kit contains Quick CIP, a heat-labile recombinant version of calf intestinal alkaline phosphatase (CIP) purified from a recombinant source. Quick CIP nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. It also hydrolyses ribo- and deoxyribonucleoside triphosphates (NTPs and dNTPs). Quick CIP is useful in many applications that require the dephosphorylation of DNA or RNA ends. In cloning, dephosphorylation prevents re-ligation of linearized plasmid DNA. The enzyme can quickly dephosphorylate 5´ protruding, 5´ recessed, and blunt ends in just 10 minutes. Quick CIP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.

Quick CIP is completely and irreversibly inactivated by heating it at 80°C for 2 minutes, unlike wild type CIP, which is heat-inactivatable. This makes removal of Quick CIP prior to ligation or end-labeling unnecessary.

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

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X
Quick CIP-20

Advantages and Features

Features

  • Rapid and irreversible heat inactivation eliminates unwanted activity
  • Improved storage stability versus native enzyme
  • Faster reaction setup (no supplemental additives like zinc required) and shorter incubation time
  • 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 (Quick CIP removes 5´- and 3´- phosphates from DNA, RNA and dNTPs )
  • Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysis
  • Recombinant for purity, consistency and value

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

Storage Temperature

-20°C

Notes

  1. Quick CIP, as are most alkaline phosphatases, is a Zn2+ and Mg2+-dependent enzyme. Our formulation has tightly bound zinc atoms in the active center and does not require supplemental zinc or other additives.
  2. Quick CIP is also active in 1X NEBuffers 1.1, 2.1, 3.1 as well as NEBuffers 1, 2, 3 and 4.
  3. Quick CIP activity is enhanced in the presence of monovalent salts.
  4. Quick CIP is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs.
  5. The Quick CIP activity is decreased in the presence of reducing agents (DTT, β-mercaptoethanol).
  6. The source for Quick CIP is a Pichia pastoris clone that carries the calf intestinal alkaline phosphatase gene from calf intestinal mucosa.
  7. The storage conditions for Quick CIP are 25 mM Tris-HCl, 1 mM MgCl2, 0.1 mM ZnCl2, 50% Glycerol, pH 7.5 @ 25°C.
  8. Quick CIP can be heat inactivated for 2 minutes at 80°C.
  9. Quick CIP is a homodimer. The molecular weight of the monomer is 56 kDa.

References

  1. Weissig, H. et al. (1993). Biochem. J. 290, 503-508.
  2. Manes, T (1998). J. Biol. Chem.. 273, 23353-23360.

FAQs

  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 Quick Dephosphorylation Kit worked?
  3. What phosphate groups are removed by rSAP, CIP, Quick CIP, or AnP?
  4. Will Quick CIP from the Quick Dephosphorylation Kit work in restriction enzyme NEBuffers?
  5. Does the DNA need to be purified after treatment with the Quick Dephosphorylation Kit?
  6. What is the effect of metal chelators, inorganic phosphate and phosphate analogs on Quick CIP activity?
  7. What is the effect of monovalent salts on Quick CIP activity?
  8. What is the effect of reducing agents on Quick CIP activity?
  9. Will Quick CIP, rSAP, CIP, or AnP dephosphorylate proteins?
  10. Can Quick CIP be heat inactivated?

Protocols

  1. Protocol for Dephosphorylation of 5´-ends of DNA using Quick Dephosphorlyation Kit (M0508)

Selection Charts

Interactive Tools

Application Notes

Certificate of Analysis

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]

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]

Safety Data Sheet

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