Shrimp Alkaline Phosphatase, Recombinant (rSAP) is a heat-labile hydrolase enzyme produced in Pichia Pastoris that removes phosphate groups nonspecifically from 5’ ends of nucleic acid phosphomonoesters and proteins. This activity is most commonly utilized in molecular cloning to prevent self-ligation of linearized plasmid DNA and in 5’ end-labeling to facilitate the replacement of unlabeled phosphates with labeled phosphate groups. rSAP also prepares PCR products for DNA sequencing or SNP analysis, by dephosphorylating unincorporated dNTPs that would otherwise interfere with enzymatic reactions.
AMRESCO’s rSAP may be directly added to restriction enzyme digests and is conveniently inactivated 100% by heating at 65°C. This eliminates the need for vector purification, a step that is necessary when using alkaline phosphatases isolated from other sources, such as E. coli and calf intestine. rSAP works well in common buffers and does not require supplemental zinc or other additives.
AMRESCO’s Shrimp Alkaline Phosphatase, Recombinant (rSAP) is highly stable at room temperature, yet completely inactivated by brief heat treatment. (A) rSAP was tested for activity at several time points after up to 81 days of storage at room temperature. Even with prolonged room temperature storage, rSAP maintained 96% of its original activity. (B) Samples of rSAP were heated to 75°C or 65°C for up to 10 minutes, with activity measured at several time points. After just 2 minutes at 75°C and 5 minutes at 65°C, rSAP activity declined to 0%.
Shrimp Alkaline Phosphatase, Recombinant (rSAP) treatment reduces empty vector background in molecular cloning. Linearized pUC19 was treated with rSAP or water prior to ligation with an insert. The rSAP was irreversibly heat-inactivated and the samples were used without any purification steps before the ligation reaction.
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