Shrimp Alkaline Phosphatase (ArcticZymes)
Shrimp Alkaline Phosphatase (ArcticZymes)
Brand: ArcticZymes
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Shrimp Alkaline Phosphatase (ArcticZymes)
Developed at ArcticZymes, first launched in 1993. Originates from the Arctic Shrimp (Pandalus borealis), from 2010 it has been produced as a recombinant version (rSAP). rSAP is better suited than native SAP (nSAP) for newer, more sensitive technologies.
Shrimp Alkaline Phosphatase (rSAP) has become one of today’s most-selling DNA modifying enzymes due to the added convenience through a complete heat inactivation. While most other alkaline phosphatases (from E. coli or Calf intestine) must be removed by extraction procedures, rSAP is completely inactivated after 15 minutes at 65°C. rSAP works well in common buffers without the requirement for other additions.
Main advantages with rSAP
- 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
Properties
Specifications
Unit definition: One Unit will convert 1 µmol of p-nitrophenyl phosphate per minute to nitrophenol and phosphate at 37ºC and pH 10.4 in 0.1 M glycine buffer, 1 mM each of ZnCl2 and MgCl2 and 6 mM 4-nitrophenyl phosphate.
This Unit definition makes 1 Unit of SAP equivalent to 5 to 40 Units of Antarctic Phosphatase (New England Biolabs), 1.3 Units APex phosphatase or 35 Units NTPhos phosphatase (Epicentre Biotechnologies). SAP is therefore the most processive alternative.
- Specific activity: >2 000 Units/mg.
- Purity: DNase activities not detected.
- Concentration: Minimum 10 000 Units/ml, available up to 30 000 Units/ml.
- Batch sizes: 25 – 35 million Units / batch.
Stability
Stable at -20ºC in storage buffer (25 mM Tris-HCl pH 7.6, 5 mM MgCl2, glycerol 50%). At room temperature, >95% activity remains after 90 days.
Still, the enzyme is completely inactivated after 5 minutes at 65°C. At 75°C, SAP is completely inactivated after only 1 minute. In a standard thermocycler, the process of heating from 37 to 95 and back to 37°C is sufficient to completely inactivate SAP.
Protocol including restriction cutting
In this protocol, SAP is present during restriction cutting, so the termini are dephosphorylated as soon as they are formed. In this protocol, the minimum effective amount of SAP is proportional to the amount of restriction enzyme added (i.e. the rate of terminus formation). In the simple sense, use at least 0.1 U SAP per Unit restriction enzyme, and proceed to complete cutting. The amount of restriction enzyme may differ from the protocol below, please use amounts recommended by the supplier.
- 1 µg plasmid
- 5 Unit restriction enzyme
- 5 µl 10x restriction enzyme buffer
- 1 Unit SAP
- dH2O to 50 µl
Incubate at 37°C for 1 hour, inactivate as recommended for the restriction enzyme used. Proceed to ligation protocol.
Quick dephosphorylation of cut plasmid
Efficient dephosphorylation can be achieved in short time using high amounts of SAP. When restriction cutting is complete, simply add 5 U SAP per µg vector to your restriction mix and incubate for further 5 min at 37°C. Inactivate as recommended for the restriction enzyme. Proceed to ligation protocol.
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