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DNA Modifying Enzymes and Cloning >
Phosphatases and Sulfurylases >
Antarctic Phosphatase |
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Prices are in US dollars and valid only for US orders.
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- 100% heat inactivated in 5 minutes at 65°C
- Ligate without purifying vector DNA
- Isolated from a recombinant source
- Removes 5' phosphates from DNA, RNA, rNTPs and dNTPs
- Prevents recircularization of cloning vectors
- Supplied with 10X Reaction Buffer
Description:
Antarctic Phosphatase catalyzes the removal of 5´ phosphate groups from DNA and RNA. Since phosphatase-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.
10 units of each phosphatase were incubated under recommended reaction conditions (including DNA) for 30 minutes and then heated at 65°C. Remaining phosphatase activity was measured by p-nitrophenyl-phosphate (pNPP) assay.
Source:
An E. coli strain that carries the TAB5 AP gene, originally cloned in plasmid pNI (2), recloned in plasmid pEGTAB7-4.1(3).
Applications:- Removing 5´ phosphoryl groups from nucleic acids
- Preparing templates for 5´end labeling
- Preventing fragments from self-ligating
- Removal of dNTPs and pyrophosphate from PCR reactions
Reagents Supplied:
Antarctic Phosphatase Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
65°C for 5 minutes
Reaction & Storage Conditions
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 6.0 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will dephosphorylate 1 µg of pUC19 vector DNA cut with HindIII (5´ protruding ends), EcoRV (blunts ends) or PstI (5´ recessed ends) in 30 minutes at 37°C. Dephosphorylation is defined as > 95% inhibition of recirculation in a self-ligation reaction and is measured by transformation into E. coli.
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).
Concentration:
5,000 units/ml
Storage Conditions:
10 mM Tris-HCl
1 mM MgCl2
1 mM Dithiothreitol
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Antarctic Phosphatase can also be used in NEBuffers 1, 2, 3 or 4 as well as the unique NEBuffers for EcoRI and BamHI ONLY when supplemented with 10X Antarctic Phosphatase Reaction Buffer to a final concentration of 1X.
FAQs
- Which phosphatase (CIP, Antarctic or SAP) should I use?
- The number of colonies that don't contain an insert seems high, how can I tell if the Antarctic Phosphatase worked?
- Can I use the enzyme to de-phosphorylate proteins?
- Will the enzyme remove P from RNA?
- Will the enzyme remove 3’ P from DNA?
- What is the molecular weight?
- Will Antarctic Phosphatase work in restriction enzyme NEBuffers?
- Does the DNA need to be purified after the restriction digest, prior to Antarctic Phosphatase treatment?
- Does the DNA need to be purified after Antarctic Phosphatase treatment?
- Can Antarctic Phosphatase be heat inactivated?
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:
Purified free of contaminating endonucleases, exonucleases and RNases.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 25 units of Antarctic Phosphatase 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 50 units of Antarctic Phosphatase with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Transformation Assay:
pUC19 was cleaved with HindIII, HincII or PstI, each purified by Qiaprep™ spin column and resuspended in water at 0.2 mg/ml. 1 µg of each DNA was treated with 5 units of Antarctic Phosphatase. Each reaction was carried out with 1 µg of DNA in a 50 µl reaction volume with 1X Antarctic Phosphatase Reaction Buffer for 30 minutes at 37°C, followed by heat inactivation at 65°C for 5 minutes.
Ligations were performed using the NEB Quick Ligation Kit protocol with 2.5 µl (50 ng) of vector DNA directly from the heat-inactivated phosphatase reaction mix. Inserts were included in 3-fold molar excess. Either Hind III cleaved fragments of λ DNA, Hae III cleaved fragments of ΦX174 RF I DNA or Pst I cleaved fragments of λ DNA were inserted as indicated.
5 ng of each ligation was transformed into E. coli DH5-α. The equivalent of 1.0 ng were plated on LB plates that contained IPTG, X-gal and ampicillin.
RNase Assay:
Incubation of a 50 μl reaction containing 50 units of Antarctic Phosphatase with 1 μg of MS2 RNA for 4 hours at 37°C resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
References
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), 5.72.
- Rina, M. et al. (2000) Eur. J. Biochem., 267, 1230-1238.
- Guthrie, E., unpublished observations.
Reagents Sold Separately
Antarctic Phosphatase Reaction Buffer
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