Shrimp Alkaline Phosphatase (SAP)

Source: Pandalus borealis (arctic shrimp)

Applications:

1. Dephosphorylation of DNA prior to cloning.
2. PCR Clean-Up for sequencing and SNP applications.
3. Dephosphorylation of DNA prior to end-labelling using T4 PNK or OptiKinase
4. Dephosphorylation of RNA.
5. Protein dephosphorylation.

Description:

• Very high specific activity
• 100% heat-inactivated at 65°C
• Removes 5’-phosphates from DNA, RNA, dNTPs and proteins
• May be added directly to restriction enzyme digests
• No vector purification necessary
• Requires no supplemental zinc or other additives for activity
• Works directly in many different buffers
• Easy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis
  

Shrimp Alkaline Phosphatase (SAP) is a high specific activity, heat-labile alkaline phosphatase useful in many applications. Alkaline phosphatases are used for the dephosphorylation of phosphorylated ends of DNA or RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA. SAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.

Shrimp Alkaline Phosphatase has approximately the same specific activity as Calf Intestinal Alkaline Phosphatase (CIAP), and like CIAP, is active in virtually all restriction enzyme reaction buffers. Unlike CIAP, Shrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min.

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Fig. 1. Reactions were set-up using 20 units SAP, PN 70092 (excess) with or without 2 μg lambda DNA EcoR I/Hind III fragments in 20mM Tris/HCl (pH 7.5) and 5mM MgCl2 in a 100 μl total volume. The reaction was incubated at 65°C to inactivate the SAP enzyme. Aliquots from the reaction were placed on ice at selected time intervals and assayed for activity in the standard assay.

Shrimp Alkaline Phosphatase is particularly useful in preparing PCR products for applications involving sequencing⁽¹⁾, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. SAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.

USB ExoSAP-IT utilizes SAP and Exonuclease I together in a specially formulated buffer to degrade residual primers and other single-stranded DNA, as well as dNTPs, eliminating the need for alternative purification methods, such as columns, gels or magnetic separations. The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.

Properties:
Molecular Weight: Homodimer. Monomer is 55 kDa as determined by amino acid sequence.
Optimum pH: 10.4 in glycine buffer and pH 8.0 in Tris buffer.
Optimum Temperature: 37°C
Heat-Inactivation: 65°C for 15 min.
Inhibitors: 10mM DTT, 0.1% β-ME
Reaction Conditions: Active in NaCl from 10mM to 150mM, KCl from 20mM to 100mM. Requires Mg²⁺ for highest activity.

Purity:
Tested for contaminating endonucleases, exonucleases and ribonucleases.

Storage Buffer:
25mM Tris-HCl (pH 7.6), 1mM MgCl₂, 0.1mM ZnCl₂, 50% glycerol.

Assay Conditions:
The reaction mixture contains 100mM glycine, pH 10.4, 1mM MgCl₂, 1mM ZnCl₂, 10mM
p-nitrophenyl phosphate and 0.001-0.1 units of Shrimp Alkaline Phosphatase (SAP). The change in absorbance at 405 nm is monitored (3050 µl reaction volume).

Unit Definition:
One unit is the amount of enzyme which catalyzes the hydrolysis of 1 µmol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37°C.

Concentration:
1 unit/µl

Tested User Friendly^TM Functional Test:
Dephosphorylation of restriction enzyme digested plasmids (5 – 20 pmol of 5'-ends, 0.1 -
0.5 units/pmol 5'-ends). Reduces religation to < 0.5% compared to the untreated control.

PROTOCOL FOR DEPHOSPHORYLATION OF NUCLEOTIDES AND DEGRADATION OF PRIMERS PRIOR TO SEQUENCING REACTIONS OR SNP ANALYSES:

Please refer to the USB ExoSAP-IT protocol, the benchmark in PCR clean-up.

The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.

Functionally Tested 10X SAP Reaction Buffer (Included, PN 70103):
200mM Tris-HCl (pH 8.0), 100mM MgCl₂.

Functionally Tested SAP Dilution Buffer (1 ml included, PN 72761):
50mM Tris-HCl (pH 8.0).

References:

1. RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, (No.1), United States Biochemical Corporation, Cleveland, OH.
2. WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
3. HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
   
   

Storage:
Shipped on dry ice. Store at -20°C.