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Science, at bottom, is really anti-intellectual. It always distrusts pure reason, and demands the production of objective fact. ~H.L. Mencken, Minority Report: H.L. Mencken's Notebook, 1956
Shrimp alkaline phosphatase or SAP, is an enzyme which catalyzes the dephosphorylation of 5’ phosphates from nucleic acid dephosphorylating RNA or DNA ends.
Note that PCR products and also primers do not have phosphatases on their free ends, and therefore need to be phosphorylated prior to cloning if you are going to use a de-phosphorylated vector!
SAP is temperature unstable are therefore you can easily deactivate this enzyme (compared to CIP!). SAP is deactivated with 65°C water bath incubation for 15 minutes.
You get virtually no reaction. SAP requires at least 10mM MgCl2 (but optimal enzyme activity of shrimp alkaline phosphatase is at 20-25mM MgCl2) and it works well at pH 8.0.
To dephosphorylate 1 pmol of DNA ends (2.5 ug of 3 Kb plasmid), for 1hr at 37°C you need:
0.1 units of SAP for 5' overhangs, 0.2 units for blunt and 0.5 units for 3' over hangs.
Enzyme comes 1 unit per ul, Thus to be safe you can use approx.0.5 units for 2 ug.
TIPS: SAP Enzyme can be added directly to restriction digest! Thus the whole proceeding including restriction enzyme digestion, dephosphorylation, enzyme inactivation, ligation or 5’ end-labeling can be performed in one single tube by just adding the appropriate reagents. This will allow you to save time and be efficient!
Discuss and post questions about it in the Shrimp Alkaline Phosphatase and DNA Cloning Forum !
Restriction Enzyme Digestion - all you need to know !
Genomic DNA Isolation Protocol
DNA Transformation - Contains History of DNA
Find other protocols at our DNA Protocols external resource directory or see our DNA protocols.
Related:
Visit DNA Bioinformatics.
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