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Product Introduction
T4 polynucleotide kinase (T4PNK) can catalyze the transfer of ATP γ-site phosphate groups to the 5 '-hydroxyl terminal of oligonucleotide chains (double-stranded or single-stranded DNA or RNA) and 3' -monophosphate nucleosides. T4 polynucleotide kinase also has 3 'phosphatase activity, hydrolyzing off 3' -phosphate groups from the 3 '-phosphate terminus of oligonucleotides, deoxy3' -nucleoside monophosphate, and deoxy3 '-nucleoside diphosphate. After modification, the 3 'phosphatase activity of the enzyme was lost, but all kinase activity was still retained.
Application
1. Phosphorylation of the end of DNA or RNA5′ in order to carry out the linking reaction.
2. End markers of DNA or RNA, used as probes and DNA
sequencing
3. The 5′ mononucleotide at the 3′ end is phosphorylated to prepare pNp substrate for addition to the 3′ end of DNA or RNA
4. Label the 5′ end of the 3′ end oligonucleotide with a phosphate group
Precautions
(1) 1×T4 polynucleotide kinase reaction buffer: 70mMTris-HClpH 7.6, 10mMMgCl2, 5mMDTT, incubated at 37℃.
(2) Thermal inactivation: heating at 65℃for 20 minutes.
(3) In the radiolabelling experiment, 1×T4 polynucleotide kinase reaction buffer, 50pmol of γ-[32P]ATP and 20 units of enzyme were incubated at 37℃for 30 minutes.
(4) T4 polynucleotide kinases require ATP to be active, but in order to be suitable for high-activity radiolabelling reactions, the reaction buffer provided with the enzyme does not contain ATP. Therefore, when phosphorylating and modifying nucleic acids, please add the final concentration of 0.5~1mMATP alone.
(5) To improve the phosphorylation efficiency of the flush or 5 'dented ends, the DNA solution can be heated at 70℃for 5 minutes, then cooled on ice, and 5% (W/V) of PEG-8000 can be added before the addition of T4 polynucleotide kinase.
(6) In general, a kinase reaction is followed by a linking reaction. In this case, the T4 polynucleotide kinase reaction was incubated in the ligase reaction buffer at 37℃for 30 minutes. The reaction product can be directly connected without changing the buffer and thermal inactivation. If other DNA fragments need to be dephosphorylated at the time of binding, T4 polynucleotide kinase needs to be thermally inactivated prior to the binding reaction.
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
