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Home > Gene Recombination / Cloning > T4 DNA Ligase

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.
Cat. No. Size Price Quantity  
TB10006A 25000 U USD 60
TB10006B 5 x 25000 U USD 240
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

  • T4 DNA Ligase 250 unit/μl
  • 10× T4 DNA Ligase Buffer

Description

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.

Applications

  • Cohesive and blunt end DNA ligation
  • DNA fragment insertion into vectors
  • Linker or adaptor ligation to a DNA fragment

Source

T4 DNA Ligase is prepared from an E. coli strain carrying the plasmid that enable highly expression of T4 DNA.

Activity Determination

One unit of T4 DNA Ligase is defined as the amount of enzyme required to catalyze the ligation of greater than 90% of λ DNA-HindIII fragments in 20 μl at 16°C in 30 minutes. One unit of this enzyme corresponds to 0.008 Weiss units.

10× T4 DNA Ligase Buffer

  • 660 mM Tris-HCl (pH 7.6 @ 25°C)
  • 66 mM KCl
  • 100 mM DTT
  • 1 mM ATP

Storage Buffer

  • 10 mM Tris-HCl (pH 7.5 @ 25°C)
  • 50 mM KCl
  • 1 mM DTT
  • 0.1 mM EDTA
  • 50% Glycerol

Inhibition

  • 50% inhibition by greater than 150 mM NaCl
  • Other inhibitors include 0.2 M K+, Cs+, Li+, NH4+ and 1 mM spermine

Heat Inactivation

65ºC for 10 min

Protocol

Set up the following reaction in a microcentrifuge tube on ice.

Component 20 μl Reaction
10× T4 DNA Ligase Buffer 2 μl
Vector DNA (5000 bp) 30 ng (0.02 pmol)
(=0.02 pmol ×  1000 ng  ×  4500 bp )
3 pmol 1000 bp
Insert DNA (800 bp) 16 ng (0.06 pmol)
(=0.06 pmol ×  1000 ng  ×  800 bp )
3 pmol 1000 bp
Nuclease free water To 20 μl
T4 DNA Ligase 1 μl
 
  • Recommended molar ratio of Vector DNA to Insert DNA is 1:3.
  • Sticky ends are ligated effectively at 16°C for 3 hours or 4–8°C overnight, while blunt-end ligations generally are efficient at 15–25°C for 4–18 hours.

Quality Control

Absence of Exonuclease Activity: Incubation of 250 U of the sample with 1 µg λ-HindIII DNA for 16 hours at 37ºC resulted in no smearing of bands on agarose gels.

Absence of Endonuclease Activity: Incubation of 250 U of sample with 1 µg of supercoiled pBR322 DNA in 1× T4 DNA Ligase Buffer for 16 hours at 37ºC resulted in no detectable conversion to relaxed or linear forms by agarose gel electrophoresis.

Physical Purity: >95% pure by SDS-PAGE.

Notes

  • ATP is an essential cofactor for the reaction. Store the 10× T4 DNA Ligase Buffer in small aliquots at –20°C to minimize degradation of the ATP and DTT.
  • The DTT in the 10× T4 DNA Ligase Buffer may precipitate upon freezing. If this occurs, vortex the buffer until the precipitate is in solution.

References

  • Weiss, B., Jacquemin-Sablon, A., Live, T.R., Fareed, G.C. and Richardson, C.C. (1968) J. Biol. Chem., 243, 4543-4555.
  • Wilson, G.G. and Murray, N.E. (1979) J. Mol. Biol., 132:471-491.
  • Engler, M.J. and Richardson, C.C. (1982) In: The Enzymes, Boyer, P.D., Ed., Academic Press, New York, NY, 3-29.
  • Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd Ed.). 1.53-1.73.

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