Description
TSAP Thermosensitive Alkaline Phosphatase removes 5´ phosphate groups from DNA, thus preventing the recircularization and religation of linearized cloning vector DNA during ligation. TSAP is effective on 3´ overhangs, 5´ overhangs and blunt ends. The enzyme is also useful for preparing DNA for 5´ end-labeling by removing existing phosphate groups from the 5´ end.
Because TSAP is irreversibly inactivated by heating at 74°C for 15 minutes, the need for a DNA cleanup step before a ligation reaction is eliminated. TSAP is fully active in all Promega restriction enzyme reaction buffers, streamlining the restriction digestion, dephosphorylation and ligation reaction workflow. TSAP Thermosensitive Alkaline Phosphatase is provided with Promega MULTI-CORE™ Buffer.
Reference
- Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Units listed are MBU (molecular biology units).
Storage Buffer: 10mM Tris (pH 7.5 @ 25°C), 100mM NaCl, 50% glycerol.
Source: Bacteria.
QC Tests: Activity, SDS-PAGE/purity, endonuclease, DNase, RNase, blue/white cloning assay.
Unit Definition: One MBU is defined as the amount of enzyme required to dephosphorylate 1μg of pGEM® DNA with 3´ overhangs in 15 minutes at 37°C in a 50μl reaction using any Promega restriction enzyme buffer (except F). Dephosphorylation is defined as preventing >95% of the plasmid from ligating (analyzed by agarose gel electrophoresis).
Comparison of Alkaline Phosphatases.
| TSAP* | SAP** | CIAP* | |
| Heat Inactivated | Yes | Yes | No |
| Inactivation Temperature | 74°C | 65°C | N/A |
| Incubation Time | 15 minutes | 15 minutes | 2 × 30 minutes |
| Special Buffer Required/Recommended | No | Yes | Yes |
| Active in All Promega Restriction Enzyme (RE) Buffers | Yes | Partially | No |
| Units Required in Different RE Buffers | 1–2 | 10 | N/A |
| Blue/White Cloning Qualified | Yes | Yes | Yes |
TSAP and SAP offer the convenience of heat inactivation. Only TSAP does not require a special buffer and is active in all Promega restriction enzyme buffers, making it the most convenient and cost-effective choice.
*TSAP=Thermostable Alkaline Phosphatase; CIAP = Calf Intestinal Alkaline Phosphatase.
**SAP = Shrimp Alkaline Phosphatase. Shown for comparison only; product not offered by Promega.
N/A = Not applicable.
Protocols
What’s in the box?
Size: 100 units
| Item / Catlog no: M9910 | Part # | Size | Available Separately |
| TSAP Thermosensitive Alkaline Phosphatase | M991A | 1 × 100u | |
| MULTI-CORE™ 10X Buffer | R999A | 1 × 0.25ml | View Product |
SDS
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.
Storage Conditions
