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Cat. No.: T4PKK-50 (for 50T)

Cat. No.: T4PKK-200 (for 200T)

Description

T4 Polynucleotide Kinase Kit, also known as T4 PNK Kit, is based on the kinase activity of T4 polynucleotide kinase (T4 PNK). This kit can be used to catalyze the transfer of phosphate groups from ATP to the 5’ hydroxyl groups of single-stranded or double-stranded DNA, RNA, oligonucleotides, or single nucleotides with a 3’ phosphate group, achieving 5’ end phosphorylation. Additionally, based on the phosphatase activity of T4 PNK, it can catalyze the dephosphorylation of 3’ phosphates in single-stranded or double-stranded DNA, RNA, and oligonucleotides.

T4 PNK is a polynucleotide 5’ hydroxyl kinase that can catalyze the transfer of the γ-phosphate group from ATP to the 5’ hydroxyl groups of single-stranded or double-stranded DNA, RNA, oligonucleotides, or single nucleotides with a 3’ phosphate group. Other NTPs can also produce the same reaction: 5’-OH + NTP → 5’-P + NDP. This phosphorylation reaction is reversible. In the absence of ATP and the presence of ADP, T4 Polynucleotide Kinase can exhibit 5’ phosphatase activity, catalyzing the transfer of the 5’ phosphate groups from single-stranded or double-stranded DNA, RNA, oligonucleotides, or single nucleotides with a 3’ phosphate group to ADP, forming ATP. Other NTPs can also produce the same reaction: 5’-P + NDP → 5’-OH + NTP (optimal pH around 6.4). When both ATP and ADP are present in appropriate amounts, T4 Polynucleotide Kinase can catalyze the exchange reaction between the 5’ phosphate groups of single-stranded or double-stranded DNA, RNA, oligonucleotides, or single nucleotides with a 3’ phosphate group and the γ-phosphate group of ATP. Other NTPs can also produce the same reaction: 5’-P + NTP + NDP → 5’-P + NDP + NTP. T4 Polynucleotide Kinase also has 3’ phosphatase activity, catalyzing the dephosphorylation of 3’-phosphorylated polynucleotides: 3’-P → 3’-OH + Pi (optimal pH around 5.9). The kinase activity of T4 Polynucleotide Kinase is near the N-terminus, while the phosphatase activity is near the C-terminus.

Application

For 5’ end labeling of oligonucleotides, DNA, or RNA, used as probes for Southern, Northern, EMSA, etc., as markers for gel electrophoresis, DNA sequencing primers, PCR primers, etc.; to phosphorylate the 5’ ends of oligonucleotides, DNA, or RNA to ensure smooth subsequent ligation reactions; to catalyze the 5’ phosphorylation of single nucleotides with 3’ phosphorylation, allowing these nucleotides to be ligated to the 3’ ends of DNA or RNA; and to remove 3’ phosphate groups.

Storage 

Store at -20ºC, effective for at least one year.

Precautions

• The 10X T4 PNK Buffer provided in this kit already contains ATP, so no additional ATP is needed.
• PCR products should be appropriately purified before 5’ phosphorylation with this product.
• DNA produced by restriction enzyme digestion can often be directly used for 5’ phosphorylation and 3’ dephosphorylation without purification. Although reaction efficiency may vary, it will not affect blunt-end cloning. For high-throughput sequencing library preparation, consider whether purification is necessary before subsequent reactions.
• The buffer in this kit is well compatible with T4 DNA ligase. If used for subsequent T4 DNA ligase ligation reactions, purification to remove the buffer is not required.
• This product is for scientific research use only by professionals. It is not intended for clinical diagnosis or treatment, nor for use in food or drugs, and should not be stored in a residential area.
• For your safety and health, please wear a lab coat and disposable gloves while handling.

Related:

• T4 Polynucleotide Kinase Kit
• T4 Polynucleotide Kinase
• T4 Polynucleotide Kinase (3' phosphatase minus)