Phosphatase, Alkaline, Shrimp, Recombinant, BioAssay™

Dry Ice

Molecular Biology Grade

Recombinant Shrimp Alkaline Phosphatase (rSAP) is a high specific activity, heat-labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis (arctic shrimp). rSAP is useful in many molecular biology applications such as the dephosphorylation of phosphorylated ends of DNA or RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents relegation of linearized plasmid DNA. rSAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.
rSAP 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, rSAP is completely and irreversibly inactivated by heating reactions at 65°C for 15 min.
rSAP is particularly useful in preparing PCR products for applications involving sequencing, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. rSAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.

Kit Components:
P4071-05B1: Phosphatase, Alkaline, Shrimp, Recombinant (rSAP) (1unit/ul), 1x500u
P4071-05B2: Shrimp Alkaline Phosphatase (SAP) Reaction Buffer (10X), 1x1ml
P4071-05B3: Shrimp Alkaline Phosphatase Dilution Buffer, 1x500ul

rShrimp Alkaline Phosphatase is particularly useful in preparing PCR products for applications involving sequencing, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. rSAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.
The enzymatic activity and heat inactivation profile of recombinant is identical to nSAP.

• 100% heat-inactivated in 5 min at 65°C
• Significantly improved storage stability at lower temperatures
• Very high specific activity
• 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 direct in many different buffers
• Easy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis

Molecular Weight: Homodimer. Monomer is 55kD 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% b-ME
Reaction Conditions:
Active in sodium chloride, potassium chloride. Requires Mg2+ for highest activity.

Purity:
Tested for contaminating endonucleases, exonucleases and ribonucleases.

Storage Buffer:
25mM Tris-HCl, pH 7.5, 1mM MgCl2, 50% glycerol.

Assay Conditions:
The reaction mixture contains 100mM glycine, pH 10.4, 1mM magnesium chloride, 10mM p-nitrophenyl phosphate and 0.001-0.1 units of P4071-05B1, recombinant Shrimp Alkaline Phosphatase (rSAP). The change in absorbance at 405nm is monitored (3050ul reaction volume).

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

Concentration:
1 unit/ul

Functional Assay:
Dephosphorylation of restriction enzyme digested plasmids (5-20 pmol of 5'-ends, 0.1-0.5units/pmol 5'-ends). Reduces religation and transformation to <0.5% compared to an untreated control.

Storage and Stability:
For long-term storage, aliquot and store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.