New England Biolabs, M0289S, Antarctic Phosphatase

Antarctic Phosphatase (AnP) is a heat labile alkaline phosphatase purified from a recombinant source. Related Categories Phosphatases,, RNA Modification Applications Dephosphorylation,, RNA Cloning Specification 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). FAQ Q: Which alkaline phosphatase, Quick CIP, rSAP or Antarctic Phosphatase works best? A: Quick CIP (NEB #M0525) is the superior choice because it offers a quick protocol, does not require any additives and has high specific activity. Quick CIP is preferred over Antarctic Phosphatase because it does not require added zinc or any other co-factors in the reaction buffer. As a result, Quick CIP can be added directly to restriction enzyme digests. Additionally, after heat-inactivation of this enzyme, it is not necessary to purify vector DNA prior to the ligation reaction Q: The number of colonies that don't contain an insert seems high, how can I tell if the Antarctic Phosphatase worked? A: The following controls can be used to diagnose this problem: 1. Uncut vector to check cell viability and test the antibiotic resistance of the plasmid. Plate on media and media plus antibiotic. 2. Vector cut and not ligated to check for uncut vector. Gel purification of the cut vector (we recommend the Monarch DNA Gel Extraction Kit, NEB# T1020) may be required to remove the last 0.1% of uncut vector. 3. Vector cut and ligated: to check for intact ends and ligation conditions. 4. Vector cut, phosphatased and ligated to check for dephosphorylation; should be 3-5% of the vector cut and ligated. Ligation conditions should also be checked (FAQs available for T4 DNA ligase, NEB# M0202). Q: Will Quick CIP, rSAP or Antarctic Phosphatase (AnP) dephosphorylate proteins? A: Quick CIP, rSAP, or AnP may be used to dephosphorylate proteins, however, NEB recommends using Lambda Protein Phosphatase (Lambda PP, NEB catalog #P0753), a dual protein phosphatase with a very broad specificity. Q: Does the DNA need to be purified after the restriction digest, prior to Antarctic Phosphatase treatment? A: In most cases Antarctic Phosphatase will work well in a reaction containing the restriction enzyme but only when supplemented with 10X Antarctic Phosphatase Reaction Buffer to a final concentration of 1X. Heat inactivate the restriction enzyme if possible. Q: Does the DNA need to be purified after Antarctic Phosphatase treatment? A: No. Heat inactivate for 2 minutes at 80°C or 5 minutes at 70°C (or as required to inactivate the restriction enzyme). If the restriction enzyme cannot be heat inactivated, the DNA should be purified prior to ligation.” Q: What is the molecular weight? A: The enzyme is 70 kDa; however the enzyme is a dimer and runs as 35kDa. Q: Can Antarctic Phosphatase be heat inactivated? A: Yes. Heat inactivate for 2 minutes at 80°C or for 5 minutes at 70°C. Alternatively, heat as required to inactivate the restriction enzyme. Q: What phosphate groups are removed by rSAP, Quick CIP, or Antarctic Phosphatase (AnP)? A: rSAP, Quick CIP, and AnP, as with any alkaline phosphatase, remove phosphates from 5´ and 3´ ends of single-stranded and double-stranded DNA and RNA and also dephosphorylates dNTPs. Alkaline phosphatases (including rSAP, Quick CIP, and AnP) have no activity on RNA 5´ cap structures. Q: Does the DNA need to be purified after a restriction digest and prior to the dephosphorylation step? A: If the enzymes used in the restriction digest can be heat inactivated, then a purification step is not needed. If the enzymes used cannot be heat inactivated, then a clean-up step between the restriction digest and the dephosphorylation step is recommended. Q: Does the DNA need to be purified after the dephosphorylation step and prior to the ligation step? A: If you use Quick Dephosphorylation Kit (NEB #M0508), rSAP (NEB #M0371) or Antarctic Phosphatase (NEB #M0289), which can be heat inactivated, then a clean-up step is not necessary. If the alkaline phosphatase used is CIP, which cannot be heat inactivated, then a clean-up step (using Monarch® PCR & DNA Cleanup Kit NEB #T1030) is necessary. Q: Are the alkaline phosphatases active in NEBuffers? A: Quick CIP (NEB #M0525), rSAP (NEB #M0371) are active in all restriction enzyme NEBuffers r1.1, r2.1, r3.1 and rCutSmart™ Buffer (NEB #B6004) and can be added directly to digested DNA.. Antarctic Phosphatase (NEB #M0289) has a strict requirement for zinc and has optimal activity at pH 6.0. Antarctic Phosphatase can 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. Q: Cloning Problem:  Too much background in the transformation step. A: In the event of having too much background in the transformation step, it is important to determine whether this is due to inefficient dephosphorylation or if it is due to undigested plasmid that has been carried over from the restriction digest. To establish the source of the background, two control samples need to be added in the transformation step: vector without ligase, and dephosphorylated vector with ligase. If the number of transformants present in sample 1 is large, this is indicative of the presence of uncut plasmid in the vector DNA. If the number of transformants in sample 1 is very low, but the number of transformants in sample 2 are high, this is indicative of inefficient dephosphorylation. If the dephosphorylation step is determined to be the problem, make sure that you add the recommended amount of phosphatase per amount of substrate recommended. Also, make sure that you incubate for the recommended time and temperature. We recommended heat-inactivating the restriction enzymes prior to the dephosphorylation step, if the restriction enzymes used can be heat-inactivated.We also recommend heat-inactivating the phosphatase prior to the ligation step. If the phosphatase used cannot be heat-inactivated, then we recommend a column cleanup of the DNA prior to the ligation step. This can be achieved by using our Monarch PCR & DNA Cleanup kit (5 ug) (NEB #T1030).