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Molecular Cloning

FastAP™ Thermosensitive Alkaline Phosphatase

Product information

Features

• Recombinant enzyme.
• Fast dephosphorylation – 10 minutes at 37°C.
• Fast and complete inactivation – 5 minutes at 75°C.
• 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 Exo I.
• Protein dephosphorylation.

Applications

• Dephosphorylation of cloning vector DNA to prevent recircularization during ligation.
• Simultaneous digestion and dephosphorylation of vector DNA.
• PCR product clean-up: nucleotide degradation prior to sequencing of PCR product.
• 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.

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. Therefore, removal of alkaline phosphatase is not required prior to ligation.

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 and 1 mg/ml BSA.

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.

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.

Figure 1. Thermoinactivation curve of FastAP™ Thermosensitive Alkaline Phosphatase at 75°C.
Bulk of vector DNA dephosphorylation reaction mixture (200 u/ml phosphatase) was incubated for 30 min at 37°C. Aliquots were heated at 75°C for time indicated in graph. The remaining activity was measured by p-NPP assay.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Dephosphorylation of DNA 5'-termini with FastAP™ Thermosensitive Alkaline Phosphatase

This protocol is suitable for removal of 3'- and 5'-phosphate groups from DNA and RNA. The protocol below is an example for dephosphorylation of ~3 kb linear vector DNA.

1. Prepare the following reaction mixture:
   

   Linear DNA (~3 kb plasmid)	1 µg (~1 pmol termini)
   10X FastAP™ reaction buffer	2 µl
   FastAP™ Thermosensitive Alkaline Phosphatase	1 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 min.
3. Stop reaction by heating at 75°C for 5 min.

Note

For efficient dephosphorylation plasmid DNA should be free of RNA and genomic DNA.

Fast Simultaneous Plasmid Vector Linearization and Dephosphorylation

1. Prepare the following reaction mixture containing:
   

   Plasmid DNA	1 µg
   10X FastDigest® buffer	2 µl
   FastDigest® Restriction Enzyme	1 µl
   FastAP™ Thermosensitive Alkaline Phosphatase	1 µl
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 10 min.
3. Stop reactions by heating at 65°C for 15 min or at 80°C for 20 min (if restriction enzyme is not inactivated at 65°C).

Note

For FastDigest® SphI (PaeI), simultaneous digestion and dephosphorylation is not recommended. Perform digestion, phenol/chloroform extract and ethanol precipitate digested DNA, dissolve DNA in 1X FastDigest® buffer and dephosphorylate.

PCR Product Clean-Up Prior to Sequencing

The clean-up reaction removes unincorporated primers and degrades unincorporated nucleotides. The resulting PCR product is ready to use for sequencing without additional purification, e.g. using column purification kits.

1. Prepare the following reaction mixture:
   

   PCR mixture (directly after completion of PCR)	5 µl
   Exonuclease I (Exo I)	0.5 µl (10 u)
   FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase (SAP)	1 µl (1 u)

2. Mix well and incubate at 37°C for 15 min.
3. Stop the reaction by heating the mixture at 85°C for 15 min.

Note

• Up to 5 µl of purified PCR products can be used directly for DNA sequencing without further purification.
• For reliable sequencing results there should not be non-specific PCR products.
• The protocol may be applied for clean-up of PCR products, generated by any thermophilic DNA polymerase or polymerase mix.
• The procedure is not recommended for downstream cloning applications.

Reference

1. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.

Radioactive Labeling of RiboRuler™ RNA Ladders by T4 Polynucleotide Kinase

The ready-to-use versions of RiboRuler™ RNA ladders can not be radiolabeled with T4 Polynucleotide Kinase.
For efficient labeling of RNA ladders it is recommended to remove 5'-phosphate groups from RNA and then phosphorylate in forward reaction using T4 Polynucleotide Kinase.
I. Dephosphorylation

1. Prepare the following reaction mixture:

   RiboRuler™ Low Range RNA Ladder or RiboRuler™ High Range RNA Ladder	8 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   10X reaction buffer for alkaline phosphatase	2 µl
   FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase	2 µl (2 u)
   DEPC-treated Water	to 20 µl
   Total volume	20 µl

2. Incubate at 37°C for 30 minutes.
3. Remove proteins from the mixture with a 20 µl aliquot of Tris-saturated (pH 8.0) phenol and chloroform mixture. Save the upper aqueous phase and extract it twice with 20 µl aliquot of chloroform.
4. Precipitate RNA by adding 1 µl of 3 M Sodium Acetate Solution, 55 µl of 96% ethanol and keep 15-30 min at -20°C. Centrifuge the mixture for 20 min at 10,000-15,000 rpm at 4°C.
5. Rinse the pellet with 20 µl cold 75% ethanol. Centrifuge 10 min at 10,000-15,000 rpm, 4°C.
6. Discard the supernatant and dissolve the air-dried pellet in 10 µl of DEPC-treated Water.

II. Labeling

1. Prepare the following reaction mixture:

   Dephosphorylated RiboRuler™ Low Range RNA Ladder or Dephosphorylated RiboRuler™ High Range RNA Ladder	1 µl 2.5 µl
   [gamma-32P]-ATP (5000 Ci/mmol,10 µCi/µl)*	5 µl (10 pmol)
   RiboLock™ RNase Inhibitor	0.25 µl (10 u)
   10X buffer A for forward reaction (supplied with T4 polynucleotide kinase)	1 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

   * If [gamma-³²P]-ATP with a high specific activity (higher than 5000 Ci/mmol) is used, the label can be diluted with ATP. Total ATP concentration should be at least 1 µM.
2. Incubate at 37°C for 30 minutes.
3. Stop the reaction by adding 1 µl of 0.5 M EDTA, pH 8.0 and extract the mixture with an equal volume of chloroform.
4. Determine the efficiency of label incorporation.
5. Load the ladder on the gel.

Dephosphorylation of Proteins

Reaction Mixture:
1X FastAP™ reaction buffer,
0.1-0.2 mg/ml of phosphoprotein,
10 u of FastAP™ Thermosensitive Alkaline Phosphatase.
Incubate at 37°C for 1 hour.

Note

• The reaction can be stopped by addition of a final concentration of 50 mM EDTA (#R1021) or by addition of a final concentration of 10 mM sodium orthovanadate (Na₃VO₄).
• The optimal incubation time and the enzyme concentration must be determined experimentally for each substrate.

Related products

• T4 Polynucleotide Kinase (T4 PNK)
• Exonuclease I (Exo I)
• T4 DNA Ligase
• T4 RNA Ligase
• Rapid DNA Ligation Kit
• CloneJET™ PCR Cloning Kit
• InsTAclone™ PCR Cloning Kit
• 6X DNA Loading Dye & SDS Solution
• Water, nuclease-free