protein

Polynucleotide Kinase T4

MBS653910

500 Units

335 USD

Enzyme

Polynucleotide Kinase T4

[Polynucleotide Kinase T4]

[PseT polynucleotide 5'-kinase and 3'-phosphatase; polynucleotide kinase]

[pseT]

E Coli infected with T4 phage

Molecular Biology Grade

Supplied as a liquid in 20mM Tris-HCl, pH 7.5, 25mM KCl, 0.1mM EDTA, 2mM DTT, 50% glycerol.

May be stored at 4 degree C for short-term only. For long-term storage, store at -20 degree C. Aliquots are stable for at least 6 months at -20 degree C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

Applications:, , 5' Labeling of nucleic acids, , Electrophoresis markers, , Transcript mapping probes, , RNA and DNA ligase substrates, , PCR primers, , DNA sequencing primers, , Hybridization probes

Unit Definition: One unit will incorporate 1 nanomole of gamma phosphate from ATP to 5'-OH DNA in 30 minutes at 37 C. Activity Assay Buffer: 100mM Tris-HCI, pH 8.0, 10mM MgCl2, 5mM DTT, O.5mM 5'-OH DNA, O.5mM ATP, 0.1 MBq/ml [g32 or g33p]-ATP. Inhibitors: Metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 5OmM. Inactivation: Heat at 70 degree C for 10 min or by the addition of EDTA. Forward Reaction Buffer (10X): Supplied as a liquid in 500mM Tris-HCI, pH 7.6, 25 degree C, 100mM MgCl2, 50mM DTT, 1mM Spermidine. Exchange Reaction Buffer (10X): Supplied as a liquid in 0.5M Imldazole-HCl, pH 6.4, 25 degree C, 0.18M MgCl2, 50mM DTT, 1 mM Spermidine, 1mM ADP. PEG Solution: Supplied as a liquid in 24% PEG 6000. Quality Control: 1. Endodeoxyribonuclease Assay. 2. Exodeoxynbonuclease Assay. 3. Ribonuclease Assay. 4. Functional Assay

Cloning; Cloning-Enzymes

T4 polynucleotide kinase catalyzes the transfer of the terminal phosphate of ATP (gamma-orthophosphate) to the 5'-hydroxyl termini of ribo- and deoxyribonucleotides. In the presence of ADP the enzyme also catalyzes an exchange reaction. In the exchange reaction, an excess ADP causes the enzyme to transfer the 5' terminal phosphate from phosphorylated DNA to ADP. The DNA thus dephosphorylated is then rephosphorylated by transfer of radiolabeled gamma-phosphate from gamma 32P ATP. T4 Polynucleotide Kinase is also 3'-phosphatase. It catalyzes the hydrolysis of 3'-phosphoryl groups of deoxynucleoside 3'-monophosphates, deoxynucleoside 3'/5'-diphosphates and of 3'-phosphorylpolyn ucleotides.

9632811

NP_049834.1

NC_000866.4

500 Units

335 USD

2500 Units

625