ATP-dependent recombinant T4 DNA ligase for efficient ligation of cohesive and or blunt end DNA fragments in 5 and 15 minutes respectively.
EN12, EN12-050, EN12-150
Available Options:
| SKU | Reactions | ||
|---|---|---|---|
 | EN12-150 | 150 reactions | Ask for an offer |
 | EN12-050 | 50 reactions | Ask for an offer |
Quick Ligase is an ATP-dependent recombinant enzyme isolated from Escherichia coli strain and used to clone DNA in just 5 to 15 minutes. Quick 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.
Applications:
- Ligation in just 5 – 15 minutes
- Cloning PCR products
- Cloning 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), e.g. amplified fragment length polymorphism (AFLP)
Unit definition
One (Weiss) unit of Quick Ligase catalyzes the conversion of 1 nmol of 32P from pyrophosphate into Norit-adsorbable material in 20 minutes at 37°C.
Quick Ligase (EN12) - Manual
- 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 Quick 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.
Quality control
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.