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Phosphatases and Sulfurylases >
Alkaline Phosphatase, Calf Intestinal (CIP) |
 | | | | Alkaline Phosphatase, Calf Intestinal (CIP) | | | |
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Prices are in US dollars and valid only for US orders.
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- Removes 5´ phosphates from DNA, RNA, rNTPs and dNTPs
- Prevents recircularization of cloning vectors
- Dephosphorylates serine, threonine and tyrosine residues in proteins
- Supplied with 10X Reaction Buffer
Description:
Alkaline Phosphatase 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.
Source:
Calf intestinal mucosa
Applications:- Removing 5´ and 3´ phosphoryl groups from nucleic acids
- Preparing templates for 5´end labeling
- Preventing fragments from self ligating
- Dephosphorylation of proteins
Reagents Supplied:
NEBuffer 3 (10X)
Enzyme Properties
Heat Inactivation:
No
Molecular Weight:
Theoretical: 69 kDa
Specific Activity:
3,500 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Incubate at
37°C.
1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One 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) in 1 M diethanolamine-HCl (pH 9.8) with 0.5 mM MgCl2 and 10 mM p-nitrophenylphosphate. These conditions are only used for quantitating enzyme activity.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM MgCl2
0.1 mM ZnCl2
50% Glycerol
pH 8.2 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- CIP can be used in NEBuffers 2, 3 or 4 as well as the Unique NEBuffers for EcoRI and BamHI. NEBuffer 3 gives optimum activity.
FAQs
- Which phosphatase (CIP, Antarctic or SAP) should I use?
- 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 CIP be heat inactivated?
- Why do some protocols recommend using CIP at 50°C and some recommend 37°C?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
CIP has been purified free of exonucleases, endonucleases and RNases by Finnzymes Oy (Finland), a strategic partner of New England Biolabs. Quality controls are performed at Finnzymes Oy and confirmed at New England Biolabs.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of Alkaline Phosphatase, Calf Intestinal (CIP) with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.5% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of Alkaline Phosphatase, Calf Intestinal (CIP) with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 μl reaction containing 10 units of Alkaline Phosphatase, Calf Intestinal (CIP) with 1
μg of MS2 RNA for 1 hour at 37ºC
resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
Transformation Assay:
50 µg of pUC19 in 1 ml of NEBuffer 2 was cleaved with HindIII and divided into five tubes (200 µl/tube). To the tubes 0.1, 1, 10 and 0 units of CIP were added respectively and incubated 30 minutes at 37°C. CIP was inactivated by adding 30 µl 0.1 M EDTA and incubating 1 hour at 65°C followed by phenol and chloroform extraction and ethanol precipitation. DNA was dissolved to 100 µl TE buffer. 1 µl of each DNA was then used in kinase and ligase tests. The kinase and ligase treatments were done in T4 DNA Ligase Buffer. For the insert samples, 1 µl of CIP treated (0.1 and 1.0 units) DNA and 0.5 µl (0.25 µg) of insert (λ HindIII cut) were used in the ligation. The DNA concentration in all reactions was 5 µg DNA/ml. The kinase reaction was 1 hour at 37°C and ligation was done at 16°C for 8 hours. 20 ng of each reaction was transformed to JM101 and 3 x 50 µl were plated on LB plates that contained IPTG, X-gal and ampicillin.
References
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed,), 5.72.
- Mossner, E., Boll, M. and Pfleiderer, G. (1980) Hoppe Seylers Z. Physiol. Chem., 361, 543-549.
Reagents Sold Separately
NEBuffer 3
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