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FastAP™ Thermosensitive Alkaline Phosphatase
Fast dephosphorylation of nucleic acids, nucleotides and proteins

#EF0651 Supplied with: 10X FastAP™ Buffer	1000 u (1 u/µl) (for 1000 reactions) 2x1.5 ml
#EF0652 Supplied with: 10X FastAP™ Buffer	5x1000 u (1 u/µl) (for 5000 reactions) 10x1.5 ml
Related Documents (in pdf, ~150 KB): Certificate of Analysis: #EF0651, #EF0652 MSDS (English) MSDS (English-USA) MSDS (German) Flyer

 

Features

• Fast dephosphorylation - 10 minutes at 37°C.
• Fast and complete inactivation - 5 minutes.
• Simultaneous digestion and dephosphorylation of vector DNA.
• 100% active in restriction enzyme and PCR buffers.
• One protocol for all types of DNA ends:
  • 5'-overhangs,
  • 3'-overhangs,
  • blunt-ends,
  • single nucleotides.
• PCR clean-up in conjunction with ExoI.
• Protein dephosphorylation.

Description
FastAP™ Thermosensitive Alkaline Phosphatase catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA and nucleotides. This enzyme also removes phosphate groups from proteins. FastAP™ is a novel alkaline phosphatase, which is active in all Fermentas restriction enzyme buffers as well as in PCR buffers. It dephosphorylates all types of DNA ends (blunt, 5'- and 3'-overhangs) in 10 min at 37°C. The enzyme is inactivated in 5 min at 75°C, see Fig.1 below. Therefore, removal of the alkaline phosphatase is not required prior to ligation.

Figure 1. Thermoinactivation curve of FastAP™ at 75°C.
A vector DNA dephosphorylation reaction mixture (200 units of phosphatase/ml) was incubated for 30 min at 37°C. Aliquots were heated at 75°C for various times. The remaining activity was measured by a p-NPP assay.

Applications

• Dephosphorylation of cloning vector DNA to prevent recircularization during ligation, see protocol.
• Simultaneous digestion and dephosphorylation of vector DNA, see protocol.
• PCR product clean-up: nucleotide degradation prior to sequencing of PCR product, see protocol.
• Dephosphorylation of nucleic acid 5'-termini prior to labeling with T4 Polynucleotide Kinase.
• Other applications where dephosphorylation of DNA and RNA substrates is necessary.
• Protein dephosphorylation.

Quality Control
Absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for dephosphorylation of 5'-termini (overhanging, recessed, blunt) of DNA.

Concentration
1 u/µl

Source
E.coli cells with a cloned bacterial AP gene.

Definition of Activity Unit
One unit is amount of the enzyme required to dephosphorylate 1 µg of linearized pUC57 DNA 5'-termini in 10 min at 37°C in FastAP™ buffer.

Storage Buffer
The enzyme is supplied in: 20 mM HEPES-NaOH (pH 7.4), 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.1% Triton X-100 and 50% (v/v) glycerol.

10X FastAP™ Buffer
100 mM Tris-HCl (pH 8.0 at 37°C), 50 mM MgCl₂, 1 M KCl, 0.2% Triton X-100, 10 mM 2-mercaptoethanol and 1 mg/ml BSA.

Inhibition and Inactivation

• Inhibitors: metal chelators.
• Inactivated by heating at 75°C for 5 min.

Note

• Binding of FastAP™ Thermosensitive Alkaline Phosphatase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 65°C for 10 min and chil on ice prior to electrophoresis.
• FastAP™ Thermosensitive Alkaline Phosphatase is active in all restriction enzyme buffers and may be added directly to digested DNA. Heat inactivation of the restriction enzyme before dephosphorylation reaction is not necessary.

Related Products

T4 Polynucleotide Kinase Exonuclease I, E.coli T4 DNA Ligase T4 RNA Ligase Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit CloneJET™ PCR Cloning Kit InsTAclone™ PCR Cloning Kit 6X Loading Dye & SDS Solution Water, nuclease-free

Updated kovo 18, 2008 09:40