Thermo Scientific T4 DNA Ligase catalyzes the connection between the side-by-side 5-phosphate and 3-hydroxyl ends of double helix DNA or RNA Formation of phosphodiester bonds. The enzyme can repair single-stranded gaps in double-helix DNA, RNA, or DNA/RNA hybrids. It can also bind DNA fragments with sticky or blunt ends, but is inactive against single-stranded nucleic acids.

T4 DNA ligase requires ATP as a cofactor.

Product Advantages

• Active in Themo Scientific restriction enzymes, PCR and RT buffers (when supplemented with ATP)
• Fast —Cohesive end ligation in 10 minutes at room temperature
• Supplied with PEG solution for efficient blunt-end ligation

Applications

• Cloning DNA fragments generated by restriction enzymes
•Clone PCR products
• Attach double-stranded oligonucleotide linkers or adapters to DNA
• Site-directed mutagenesis
• Amplify fragment length polymorphisms (AFLP)
• Ligase-mediated RNA detection (seeReference 3)
• Gap repair in duplex DNA, RNA, or DNA/RNA hybrids
• Self-circularization of linear DNA.

Includes

• T4 DNA Ligase
• 10X T4 DNA Ligase Buffer
• 50% PEG Solution

Notes

• Binding of T4 DNA ligase to DNA may cause band shifts in agarose gels. To avoid this, use 6X DNA Loading Dye & SDS Solution at 70°C Incubate samples for 5 minutes at 65°C or 10 minutes at 65°C, cool on ice before electrophoresis.
•During the transformation process, the volume of the ligation reaction mixture should not exceed 10% of the volume of the competent cells.
•Before electroporation, remove T4 DNA ligase from the ligation mixture using spin columns or chloroform extraction. The extracted DNA can be further ethanol precipitated.