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T4 DNA Ligase

B buffer for 100% activity Blue/white certified FastDigest buffer for 100% activity G buffer for 100% activity High concentration available LO certified O buffer for 100% activity Recombinant enzyme R buffer for 100% activity Tango buffer for 100% activity Thermal inactivation at 65°C in 10 min

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA.
  
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Thermo Scientific 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 (see References 1, 2).

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.
  
Storage Condition-20 C
Shelf Life0 Months
HazardousNo
10X T4 DNA Ligase Buffer (#B69)400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C)
50% PEG Solution50% (w/v) polyethylene glycol 4000
Definition of Activity Unit

One Weiss unit** of the enzyme catalyzes the conversion of 1 nmol of [32PPi] into Norit-adsorbable form in 20 minutes at 37°C (4).

Enzyme activity is assayed in the following mixture: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 0.066 mM ATP, 10 mM DTT, 3.3 µM [32PPi].

** One Weiss unit is equivalent to approximately 200 cohesive-end ligation units. One cohesive-end ligation unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 minutes at 16°C in 20 µLof the assay mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 µg/mL BSA and a 5'-DNA termini concentration of 0.12 µM (300 µg/ml).

Inhibition and Inactivation

 

  • Inhibitors: T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM
  • Inactivated by heating at 65°C for 10 minutes or 70°C for 5 minutes

 

Molecular Weight55.3 kDa monomer
Quality Control
  • The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
  • Functionally tested for the capacity to join cohesive- and blunt-end DNA fragments.
SourceE. coli cells with a cloned gene 30 of bacteriophage T4
Storage BufferThe enzyme is supplied in:
20 mM Tris-HCl (pH 7.5), 1 mM DTT, 50 mM KCl, 0.1 mM EDTA, 0.015% ELUGENT Detergent, and 50% (v/v) glycerol.

References

  1. B. H. Pheiffer et al., Polymer-stimulated ligation: enchanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11, 7853-7871 (1983).

  2. R. Rossi et al., Functional characterization of the T4 DNA Ligase: a new insight into the mechanism of action. Nucleic Acids Res. 25, 2106-2113 (1997).

  3. A. V. Cherepanov et al.,  Binding of nucleotides by T4 DNA Ligase and T4 RNA Ligase: optical absorbance and fluorescence studies. Biophys. J. 81, 3545-3559 (2001).

  4. M. Nilsson et al., RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res. 29, 578-581 (2001).

  5. B. Weiss et al., Enzymatic breakage and joining of deoxyribonucleic acid. J. Biol. Chem. 243, 4543-4555 (1968).