|EN11-250||2500U||Ask for an offer|
|EN11-050||500 U||Ask for an offer|
T4 DNA Ligase is an ATP-dependent recombinant enzyme isolated from Escherichia coli strain used to clone DNA. T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA. It will join both blunt-ended and cohesive-ended restriction fragments of DNA, as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
- ATP-dependent recombinant DNA ligases over-expressed in E. coli.
- Available also in special formulation for very fast and efficient ligation of DNA fragments with compatible cohesive or blunt ends in 5 and 15 minutes respectively.
- Molecular cloning of PCR products or restriction fragments.
- Joining double-stranded oligonucleotide linkers or adaptors to DNA.
- Site-directed mutagenesis.
- Nick repair in duplex DNA, RNA or DNA/RNA hybrids.
- Self-circularization of linear DNA.
- LM PCR methods (Ligation Mediated PCR).
One (Weiss) unit of T4 DNA Ligase catalyzes the conversion of 1 nmol of 32P from pyrophosphate into Norit-adsorbable material in 30 minutes at 37°C.
One Weiss unit is equivalent an approximately 200 cohesive end units.
- The 10x Ligation Buffer and ATP Solution should be thawed and resuspended at room temperature.
- For blunt-end ligations, use higher quantities of both the vector and the insert DNA.
- For sticky (cohesive)-end ligations, we recommend heating both the vector and the insert DNA prior to ligation.
- The electrotransformation efficiency may be improved by heat inactivation of the T4 DNA Ligase and purifi cation of the DNA by means of a spin column purification method (EXTRACTME DNA CLEAN-UP kit or
EXTRACTME DNA GEL-OUT kit).
- We recommend using a 3 – 10 molar excess of insert DNA over vector DNA. To calculate the optimal amounts of insert DNA in a ligation reaction,use the following equation:
Example: If using 20 ng of a vector plasmid (4 kb), for a 5:1 molar ratio of insert : vector, you will require the following quantity of 1 kb insert:
- The enzyme is inhibited by >200 mM NaCl or KCl concentrations.
- Inactivate enzyme at 65°C for 10 minutes or at 70°C for 5 minutes.
The product was tested in ligation of HindIII-cut lambda DNA with a different amounts of enzyme. The product is free of unspecific DNA nucleases.
T4 DNA Ligase (EN11) – MSDS