Contents > Molecular Cloning Enzymes > Apex™ Heat-Labile Alkaline Phosphatase
Apex™ Heat-Labile Alkaline Phosphatase
Supplier: EPICENTRE® Biotechnologies
Apex™ Heat-Labile Alkaline Phosphatase
| Catalogue number | Pack size |
| AP49010 | 10 Reactions |
| AP49050 | 50 reactions |
| AP49100 | 100 reactions |
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Last modified: 2008-07-15 14:57:15
APex™ Heat-Labile Alkaline Phosphatase is a innovative enzyme preparation with improved performance over other available alkaline phosphatases.
It is derived from a recombinant source, and is thus, very pure and free from nuclease contamination. APex™ Phosphatase removes the 5'-phosphate from all types of DNA ends, including 5' protruding, blunt, and 5' recessed ends, and from RNA ends. The enzyme is irreversibly heat-inactivated by incubation at 70°C for 5 minutes.
APex™ Phosphatase can be added directly to most restriction enzyme (RE) buffers and is highly active over a broad range of temperature (up to 50°C), pH (from 5-12), and salt conditions (e.g., up to 1 M Na+, NH4+, K+, Cl- or Acetate-), and in the presence of 10% Triton® X-100. To remove the 5'-phosphate from DNA, RNA or a nucleotide, simply incubate the substrate with APex™ Phosphatase at 37°C for 10 minutes.
The same reaction conditions may be used for all types of DNA ends, including those with difficult 5' recessed ends. Buffer supplementation is not necessary in common restriction enzyme buffers, so vectors can be digested directly in these buffers. Five dATP hydrolysis units will completely dephosphorylate any type of restriction enzyme-generated 5'-end from 1µg of vector in 10 minutes at 37°C. For example, 5 dATP hydrolysis units completely dephosphorylates the ends of 1µg of pUC19 vector DNA digested with HindIII (5' protruding), HincII (blunt), or PstI (5' recessed) in 10 minutes at 37°C based on their inability to be self-ligated.
| Restriction enzyme overhangs tested | |
|---|---|
| 5'-overhang | |
| 5'-(4-base) overhang: | Hind III, EcoR I |
| 5'-(3-base) overhang: | EcoO109 I |
| 5'-(2-base) overhang: | Nde I |
| Blunt-end, no overhang: Sma I, Ssp I | |
| 5'-recessed | |
| 3'-(4-base) overhang: | Sph I, Sac I, Pst I |
| 3'-(3-base) overhang: | AlwN I |
| 3'-(1-base) overhang: | Ahd I |
Last modified: 2008-05-14 16:46:46
Applications
- Dephosphorylation of DNA vectors prior to cloning to prevent recirculization
- Preparation of 5'- nucleic acid termini for 5'-end labeling with polynucleotide kinase
- Dephosphorylation of DNA and RNA substrates
Benefits
- One simple protocol for most vector dephosphorylation reactions
- Purified from a recombinant source without nuclease contamination for improved performance
- Fast, complete and irreversible heat-inactivation or easy transition to next step; no time-consuming substrate purification with phenol:chloroform extraction
- Flexible and easy to use - add directly to most RE buffers without supplementation; active over a wide range of temperatures, pH, salts and buffers
- Active on blunt, 5'- and 3'- overhang restricted DNA ends for compatibility with any restriction enzyme and experimental design
Unit Definition:
One microlitre of APex Heat-Labile Alkaline Phosphatase dephosphorylates 1 µg of pUC19 vector DNA digested with Hind III (5' protruding), Hinc II (blunt), or Pst I (5' recessed) in 10 minutes at 37°C.
Storage Buffer:
50 mM Tris-HCl (pH 7.5), 0.1 mM Zinc Acetate, 10 mM MgCl2, 0.1 M NaCl, 50% glycerol, and 0.1% Triton® X-100.
10X Reaction Buffer:
330 mM Tris-acetate (pH 7.8), 660 mM potassium acetate, 100 mM magnesium acetate and 5.0 mM DTT.
Quality Control:
Functionally tested to meet our Cloning Quality Standard of greater than 99% inhibition of self-ligation and checked by transformation into E. coli. Free of detectable exo- and endonuclease and RNase activities.
Last modified: 2008-05-14 16:46:46
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