New England Biolabs, M0371S, Shrimp Alkaline Phosphatase (rSAP)

Shrimp Alkaline Phosphatase (rSAP) is a heat labile alkaline phosphatase purified from a recombinant source. Related Categories Phosphatases Applications Dephosphorylation,, RNA Cloning Specification Unit Definition One unit is defined as the amount of enzyme that hydrolyzes 1 μmol of p -Nitrophenyl Phosphate, PNPP ( NEB #P0757 ) in a total reaction volume of 1 ml in 1 minute at 37°C. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 25 mM Tris-HCl 1 mM MgCl2 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 5 minutes Molecular Weight Theoretical: 54 kDa Unit Assay Conditions 1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl 2 , 50 mM p -Nitrophenyl Phosphate. These conditions are only used for quantitating enzyme activity. 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 do not contain an insert seems high. How can I tell if rSAP worked? A: rSAP is a very robust enzyme and it is rare for dephosphorylation not to proceed essentially to completion. The following transformation controls can be used to diagnose this problem: Uncut vector: to check cell competency and antibiotic resistance. Vector cut and not ligated: to check for uncut vector. Gel purification (we recommend Monarch DNA Gel Extraction Kit, NEB# T1020) may be required to remove the last 0.1% of uncut vector. Vector cut and ligated: to check for intact ends and ligation conditions. Vector cut, dephosphorylated and ligated: to check for dephosphorylation. Total colony number should be <5% of the vector cut and ligated. 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 rSAP treatment? A: No, as long as rSAP is heat inactivated at 65°C for 5 minutes prior to ligation. However, if the dephosphorylation reaction is performed directly after vector DNA digestion, both restriction enzyme and rSAP should be heat-inactivated. In this case, please follow manufacturer’s recommendations to inactivate restriction enzyme. Q: How stable is rSAP in its storage buffer at various temperatures? A: Recombinant rSAP has significantly improved stability compared to native SAP. Temperature –20°C 25°C 100% activity >2 years >2 months Q: What is the effect of metal chelators, inorganic phosphate and phosphate analogs on rSAP activity? A: Metal chelators, inorganic phosphate and phosphate analogs are inhibitory. Q: What is the effect of reducing agents on rSAP activity? A: Reducing agents, DTT or β-mercaptoethanol (β-ME), may decrease rSAP activity: Reducing Agents Concentration Activity DTT 0 mM 100% 1 mM 88% 5 mM 63% 10 mM 46% 50 mM 20% β-ME 0 mM 100% 5 mM 95% 10 mM 78% 20 mM 75% rSAP activity was measured by PNPP assay in 1X rSAP Reaction Buffer supplemented with varying amounts of DTT or β-ME. Activity is shown as percent activity compared to the control, which did not contain added reducing agents. 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: Can rSAP be heat inactivated? A: Yes. Heat inactivate rSAP for 5 minutes at 65°C. 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). Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.