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Thermo Scientific™ T4 DNA Ligase


T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA.

Manufacturer: thermo scientific™  EL0011

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Catalog No. FEREL0011

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Description & Specifications

Specifications

Concentration 5U/µL
Inhibitors T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200mM.
Source E.coli cells with a cloned gene 30 of bacteriophage T4
Molecular Weight 55.3 kDa monomer
Units 1,000U

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids.

The T4 DNA Ligase requires ATP as a cofactor.

Highlights

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

Applications

  • Cloning of restriction enzyme generated DNA fragments
  • Cloning of PCR products
  • Joining of double-stranded oligonucleotide linkers or adaptors to DNA
  • Site-directed mutagenesis
  • Amplified fragment length polymorphism (AFLP)
  • Ligase-mediated RNA detection (see Reference 3)
  • Nick repair in duplex DNA, RNA or DNA/RNA hybrids
  • Self-circularization of linear DNA.

Notes

  • Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis.
  • The volume of the ligation reaction mixture should not exceed 10% of the competent cell volume in the transformation process.
  • Prior to electro-transformation, remove T4 DNA Ligase from the ligation mixture by spin column or chloroform extraction. The extracted DNA can be further precipitated with ethanol.