| 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. |
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| Catalog # | P4410 |
| Applications | Applications: |
| • 5’ Labeling of nucleic acids |
| • Electrophoresis markers |
| • Transcript mapping probes |
| • RNA and DNA ligase substrates |
| • PCR primers |
| • DNA sequencing primers |
| • Hybridization probes |
| Storage and Stability | May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 6 months at -20°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. |
| Source | E. coli infected with T4 phage |
|
| Form | Supplied as a liquid in 20mM Tris-HCl, pH 7.5, 25mM KCl, 0.1mM EDTA, 2mM DTT, 50% glycerol. |
|
| Concentration | 10u/ul |
|
| Unit Definition | One unit will incorporate 1 nanomole of gamma phosphate from ATP to 5’-OH DNA in 30 minutes at 37ºC. |
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| Inactivation | |
| Heat at 70ºC for 10 min or by the addition of EDTA. |
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| P4410A, Forward Reaction Buffer (10X) | Supplied as a liquid in 500mM Tris-HCl, pH 8.0 (25°C), 100mM MgCl2, 50mM DTT, 1mM Spermidine, 1mM EDTA. |
|
| P4410B, Exchange Reaction Buffer (10X) | |
| Supplied as a liquid in 0.5M Imidazole-HCl, pH 6.5 (25°C), 0.18M MgCl2, 50mM DTT, 1mM Spermidine, 1mM EDTA, 1mM ADP. |
|
| P4410C, PEG Solution | |
| Supplied as a liquid, 24% w/v PEG 6000. |
|
| Activity Assay Buffer | 100mM Tris-HCl, pH 8.0, 10mM MgCl2, 5mM DTT, 0.5mM 5’-OH DNA, 0.5mM ATP, 0.1MBq/ml [g32 or g33p]-ATP. |
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| Quality Control | |
| Endodeoxyribonuclease Assay | |
| No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 50u of T4 polynucleotide kinase with 1ug of pBR322 DNA in 50ul if Activity Assay Buffer for 4 hours at 37ºC. |
| Exodeoxyribonuclease Assay | |
| 0% of the total radioactivity was released into trichloracetic acid-soluble fraction after incubation of 50u of T4 polynucleotide kinase with 1ug of sonicated E. coli[3H]-DNA in 50ul if Activity Assay Buffer for 4 hours at 37ºC. |
| Ribonuclease Assay | |
| 0% of the total radioactivity was released into trichloracetic acid-soluble fraction after incubation of 50u of T4 polynucleotide kinase with 1ug of sonicated E. coli[3H]-RNA in 50ul if Activity Assay Buffer for 4 hours at 37ºC. |
| Functional Assay | |
| T4 polynucleotide kinase was tested for labeling of 5’-termini of DNA. |
|
| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
| Alternate names | EC=2.7.1.78; E. coli T4 phage; Supplied with Forward, Exchange Reaction Buffers, PEG Solution |
