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Products » All » DNA/RNA Modifying Enzymes » Phosphatases and Kinase

Phosphatases and Kinase: FastAP™ Thermosensitive Alkaline Phosphatase; Shrimp Alkaline Phosphatase (SAP); T4 Polynucleotide Kinase (T4 PNK)

Applications	Substrates	Reaction catalyzed	Reaction products	Reaction temp.	Inactivation	Phosphatase / Kinase
Phosphatase
Dephosphorylation of cloning vector DNA to avoid recircularization PCR product clean-up before sequencing	5'- and 3'-phosphorylated DNA/RNA oligonucleotides	5', 3'-OH ends of DNA/RNA oligonucleotides	Release of phosphate group	37°C	Heating at 75°C, 5 min	FastAP™ Thermosensitive Alkaline Phosphatase
Dephosphorylation of DNA or RNA 5'-ends prior to labeling with T4 PNK Dephosphorylation of DNA, RNA and oligonucleotides	dNTPs/NTPs	deoxynucleosides/ nucleosides			Heating at 65°C, 15 min	Shrimp Alkaline Phosphatase (SAP)
Dephosphorylation of proteins	Phosphorylated proteins	Dephosphorylated proteins			Heating at 65°C, 15 min	Shrimp Alkaline Phosphatase (SAP)
Kinase
Phosphorylation of PCR products prior to ligation Phosphorylation of PCR primers 5'-phosphorylation of oligonucleotide and DNA or RNA prior to ligation Labeling 5'-termini of nucleic acids Labeling of sequencing primers Labeling of DNA and RNA markers Detection of DNA modification by the [³²P]-postlabeling assay	ATP¹ DNA/RNA oligonucleotides 3'-NMP	5'-phosphorylated DNA/RNA 5'-P-oligonucleotides 3', 5'-NDP	Transfer of the gamma-phosphate from ATP¹ to the 5'-OH group of DNA/RNA, oligonucleotides, 3'-NMP. Exchange of phosphate group between 5'-P-oligo-/ polynucleotides and ATP (in the presence of ADP)	37°C	Heating at 75°C, 10 min or addition of EDTA	T4 Polynucleotide Kinase (T4 PNK)
Removal of 3'-phosphate group	3'-phosphorylated DNA/RNA 3'-P-oligonucleotides deoxynucleoside 3'-monophosphates deoxynucleoside 3'-diphosphates	3'-dephosphorylated DNA/RNA oligonucleotides deoxynucleosides	Release of 3'-phosphate group	37°C	Heating at 75°C, 10 min or addition of EDTA	T4 Polynucleotide Kinase (T4 PNK)

ss – single-stranded
ds – double-stranded
¹ – athough ATP is routinely used as a phosphate donor, CTP, UTP, GTP, d ATP, dTTP are equally effective.

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