This nice thing about this protocol is that you dont need to pipette your pcr products out of their original pcr tubes minimizing the potential for. Alkaline phosphatase alp, alkp, alpase, alk phos ec 3. After the cleanup procedure is complete the sequencing protocol of choice should be followed as described in the specific sequencing kit protocol booklet. The enzymes are heat labile and will slowly lose activity at room temperature. This enzyme is used to prevent recircularization and religation of linearized cloning vehicle dna by removing. This property simplifies most workflows involving alkaline phosphatase treatment. Shrimp alkaline phosphatase rsap new england biolabs. Prepare reaction mix in a 20 l volume according to the following table. Calf intestinal alkaline phosphatase ciap catalyzes the hydrolysis of 5. The shrimp alkaline phosphatase hydrolyzes remaining dntps from the pcr mixture which would interfere with the sequencing reaction. The improved method utilizes pnitrophenyl phosphate that is hydrolyzed by alp into a yellow colored product maximal absorbance at 405 nm.
Shrimp alkaline phosphatase rsap recombinant shrimp alkaline phosphatase rsap is a heatlabile multipurpose alkaline phosphatase that catalyzes the dephosphorylation of dna, rna, and nucleotides. Cip calf alkaline phosphatase or sap shrimp alkaline phosphatase are commonly used. Protocol for nucleic acid dephosphorylation this protocol is suitable for removal of 3 and 5 phosphate groups from dna and rna. The concentration of the enzyme stock solution is 10 ul for fermentas enzymes neb enzymes may. Sap is useful in many molecular biology applications such as the dephosphorylation of phosphorylated ends of dna or rna for subsequent use in cloning. Bacterial alkaline phosphatase bap is the most active of the enzymes, but also the most difficult to destroy at the end of the dephosphorylation reaction. Shrimp alkaline phosphatase sap is a high specific activity, heatlabile alkaline phosphatase purified from a recombinant source and originally isolated from. Shrimp alkaline phosphatase sap nonspecifically catalyzes the dephosphorylation of 5termini dna and rna phosphomonoesters, but does not degrade. Ligandbinding and metalexchange crystallographic studies on shrimp alkaline phosphatase. Shrimp alkaline phosphatase, recombinant enzprt161 enzo. This nice thing about this protocol is that you dont need to pipette your pcr products out of their original pcr tubes minimizing the potential for pcr contamination of your lab and equipment.
Sap can be used in any neb restriction enzyme buffer, but make sure buffer has at least 10 mm mgcl2. Shrimp alkaline phosphatase sap thermo fisher scientific. Shrimp alkaline phosphatase is derived from a coldwater shrimp and is promoted for being readily destroyed by heat 65c for 15 minutes. Should you use calf intestinal alkaline phosphatase cip in. Alkaline phosphatase, calf intestinal ciap protocol. What this product does content storage and stability. Protocol for fast simultaneous plasmid vector linearization and dephosphorylation 1. Shrimp alkaline phosphatase sap nonspecifically catalyzes the dephosphorylation of most phosphate monoesters, but it does not degrade diphosphate or triphosphate linkages. Alkaline phosphatase, calf intestinal cip nonspecifically catalyzes the dephosphorylation of 5. It is irreversibly inactivated by heat denaturation at 65c for 15 minutes.
If you are using blunt ends or a single enzyme to cut the vector, you will need to use a phosphatase to prevent recircularization of the vector if you are cloning in an insert. Dephosphorylation of dna using shrimp alkaline phosphatase shrimp alkaline phosphatase, cat. Fermentas en0581 4000 units, 20uul 1 unit of sap and 5 units of exoi are used to treat 10 ul pcr product typically. Public release data set information this document details the lab protocol for nhanes 20012002 data. This recombinant enzyme replaces native sap because it is much more stable at room temperature and is available at higher concentrations.
Alternatively, the linear dna was dephosphorylated with sap shrimp alkaline phosphatase, fermentas according to the manufacturers instructions. Alkaline phosphatase, calf intestinal ciap, is supplied in 10mm trishcl ph 8. Elevations in serum activity of alkaline phosphatase commonly originate from the liver and bone and are of interest in the diagnosis of hepatobiliary disorders and bone disease associated with increased osteoblastic activity. In bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. Hello everybody, i used fastap instead of shrimp alkaline phosphatase and exonuclease i for pcr purification prior to sanger sequencing. Shrimp alkaline phosphatase sap removes phosphate groups from. Would explain why i got successful ligation when neither vector nor insert were dephosphorylated and an unsuccessful ligation when the vector was treated with phosphatase. The widespread occurrence of ap in nature suggests its involvement in fundamental biochemical processes. Exonuclease i and shrimp alkaline phosphatase in buffer. New england biolabs uk ltd shrimp alkaline phosphatase rsap. Thermolabile alkaline phosphatase from northern shrimp pandalus borealis.
Oct 18, 2010 an obscure 1980 manuscript by ellen mossner and colleagues 1 first described its isolation, but the first published description of using calf intestinal alkaline phosphatase cip in gene cloning was published a while later in sambrook et als legendary 1989 manual, molecular cloning 2nd edition 2. Alkaline phosphatase, shrimp from northern shrimps p. Studies in both escherichia coli and cyanobacterium, anacystis nidulans r2, have shown increase in alkaline phosphatase activity upon phosphate deprivation. Alkaline phosphatase accounted for 80% of total released proteins. Improved stability of sap three month stability of sap at various temperatures. Alkaline phosphatase have also been found in variety of microorganism including li, pseudomonas, aerobacter and bacillus species. Usb shrimp alkaline phosphatase thermo fisher scientific. Alkaline phosphatase alp in refrigerated serum nhanes 20012002 collaborative laboratory services, l. Calf intestinal alkaline phosphatase cip new england biolabs, catalog number. The exosap protocol is the simplest way to cleanup pcr products before sequencing.
Exonuclease i shrimp alkaline phosphatase clean up of pcr. Each monomer contains five cysteine residues, two zinc atoms and one magnesium atom crucial to its catalytic function, and it is optimally active at alkaline ph environments alp has the physiological role of dephosphorylating compounds. Alkaline phosphatase ap is a hydrolase enzyme responsible for dephosphorylating molecules such as nucleotides and proteins under alkaline conditions. It is a multipurpose alkaline phosphatase that can be fully inactivated by a short heat treatment. Alkaline phosphatase alp in refrigerated serum nhanes. Resuspend 1 pmol of dna ends about 1 g of a 3 kb plasmid in nucleasefree water.
Shrimp alkaline phosphatase rsap, ligases phosphatases. Ready to load sequencing appendix ii cornell university. Exonuclease i shrimp alkaline phosphatase clean up of. Two laboratories performed this testing during 20012002. This enzyme is used to prevent recircularization and religation of linearized cloning vehicle dna by removing phosphate groups from both 5. Sap was stored for 3 months at the indicated temperatures and the remaining activity was measured using a standard pnitrophenyl phosphatase activity. Product code pack size 70092y 500 units 70092z 1,000 units 70092x 5,000 units protocol for dephosphorylation of 5ends of dna. Should you use calf intestinal alkaline phosphatase cip. Aug 28, 2012 taq dna polymerase 1u and respective buffer fermentas and datp final concentration 0. The exonuclease i removes leftover primers, while the shrimp alkaline phosphatase removes any remaining dntps. Alkaline phosphatase ap is a nonspecific phosphomonoester hydrolase that catalyzes the hydrolysis of a wide variety of organic monophosphates. Antarctic phosphatase activity is decreased in the presence of reducing agents dtt. Alkaline phosphatase reagents thermo fisher scientific. Calf intestinal alkaline phosphatase cip can be used to release phosphate groups from phosphorylated tyrosine, serine and threonine residues in proteins.
Alkaline phosphatase is also an important tool in molecular cloning and dna sequencing. Do not store in a frostfree freezer the temperature rises above 0c daily. The dualcolor alkaline phosphatase staining kit is a histochemical assay for use on cells grown in tissue culture wells or dishes. In bacteria, alkaline phosphatase is present in the periplasmic space. The widespread occurrence of ap in nature suggests its involvement in fundamental biochemical processes, however, there is no positive evidence regarding its physiological function, or the nature of. Shrimp alkaline phosphatase rsap is a heat labile alkaline phosphatase purified from a recombinant source. Heatlabile alkaline phosphatase for dephosphorylation of dna, rna and nucleotides. Because of their critical functions in eukaryotic cells, methods for measuring protein phosphatases were established at least as early as 19531. Linear dna 3 kb plasmid 1 g 1 pmol termini 10x reaction buffer for ap used in reaction 2 l fastap thermosensitive alkaline phosphatase 1 l1 u.
Each monomer contains five cysteine residues, two zinc atoms and one magnesium atom crucial to its catalytic function, and it is optimally active at alkaline ph environments. If you know the exact size and amount of that pure protein, use those numbers. Shrimp alkaline phosphatase rsap has become one of todays mostselling dna modifying enzymes due to the added convenience through a complete heat inactivation. The alkaline phosphatase assay kit is designed to measure alp activity directly in biological samples without pretreatment. This recombinant enzyme replaces native sap because it is much more stable at room temperature and is. Shrimp alkaline phosphatase rsap has become one of todays mostselling dna modifying enzymes due to the added convenience through a complete heat. The recombinant form of sap replaces the native form that has been established on the market for several years. P9088 is supplied as a solution in 50% glycerol containing 25 mm trishcl, ph 7.
Sap exonuclease i treatment protocol from martha hamblin, igd reagents needed. Shrimp alkaline phosphatase sap still the golden standard and the first heatlabile alkaline phosphatase on the market. Faster reaction setup no supplemental additives like zinc required flexible reaction conditions active in any restriction enzyme buffer, no cleanup required. Recombinant shrimp alkaline phosphatase is a multipurpose alkaline phosphatase that can be fully inactivated by a short heat treatment. Dephosphorylation of proteins alkaline phosphatase ap is a nonspecific phosphomonoester hydrolase that catalyzes the hydrolysis of a wide variety of organic monophosphates. They didnt mention that in the manual used a combination of shrimp and calf intestinal alkaline phophates from roche. Introduction and significant role of alkaline phosphatases. Storage store at 20c and keep on ice while pipeting. New england biolabs uk ltd shrimp alkaline phosphatase. In 1965 fernley and walker2 decribed the use of 4methylumbelliferyl phosphate mup as a substrate for alkaline phosphatase. Shrimp alkaline phosphatase shrimp alkaline phosphatase. The following information can be used as suggested initial conditions for dephosphorylation of proteins with cip. Shrimp alkaline phosphatase rsap grisp research solutions.
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