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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™  EL0014

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Catalog No. FEREL0014

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Description & Specifications

Specifications

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

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.