Alkaline Phosphatase (CIP) catalyzes the removal of 5´ phosphate groups from DNA, RNA, ribo- and deoxyribonucleoside triphosphates. Since CIP-treated fragments lack the 5´ phosphoryl termini required by ligases, they cannot self-ligate (1). This property can be used to decrease the vector background in cloning strategies.
Product SourceCalf intestinal mucosa
The following reagents are supplied with this product:
Advantages and Features
- Removing 5´ phosphates from DNA, RNA, rNTPs and dNTPs
- Preparation of templates for 5´end labeling
- Prevention of recircularization of cloning vectors
- Dephosphorylation of proteins
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme that hydrolyzes 1 µmol of p-nitrophenylphosphate to p-nitrophenol in a total reaction volume of 1 ml in 1 minute at 37°C (2).
1X NEBuffer 3
1X NEBuffer 3:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
1 mM DTT
pH 7.9 @ 25°C
10 mM Tris-HCl
50 mM KCl
1 mM MgCl2
0.1 mM ZnCl2
pH 8.2 @ 25°C
Molecular WeightTheoretical: 69 kDa
Specific Activity3,500 units/mg
Unit Assay Conditions1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl2, 10 mM p-nitrophenyl phosphate and enzyme. These conditions are only used for quantitating enzyme activity.
Quality Control AssaysThe 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.
- 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.
- Protein Purity (SDS-PAGE):
The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
- RNase Activity (2 Hour Digestion):
The product is tested in a reaction containing a RNA substrate. After incubation for 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
Material Safety Datasheets
- CIP is also active in CutSmart Buffer.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed,). (p5.72), Cold Spring Harbor Laboratory Press .
- Mossner, E., Boll, M. and Pfleiderer, G. (1980). Hoppe Seylers Z. Physiol. Chem.. 361
- Which alkaline phosphatase, rSAP, CIP, or Antarctic Phosphatase, works best?
- The number of colonies that do not contain an insert seems high, how can I tell if the CIP worked?
- Will CIP work in restriction enzyme NEBuffers?
- Does the DNA need to be purified after the restriction digest, prior to CIP treatment?
- Does the DNA need to be purified after CIP treatment?
- Will CIP dephosphorylate proteins?
- Can Antarctic Phosphatase remove the 3' phosphate from DNA?
- Can CIP be heat inactivated?
- Why do some protocols recommend using CIP at 50°C and some recommend 37°C?