Quick Ligase is an ATP-dependent recombinant enzyme, isolated from Escherichia coli, used for DNA cloning 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.
– Ligation in just 5-15 minutes
– Cloning of PCR products
– Cloning of restriction fragments
– Joining of 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), eg. Amplified fragment length polymorphism (AFLP)
10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 50 mM KCl, 50% (v/v) glycerol
2x Quick Ligation Buffer
132 mM Tris-HCl (pH 7.6), 20 mM DTT, 20 mM MgCl2,15% PEG 6000
The 2x Quick Ligation Buffer does not contain ATP, which must be added separately. For most applications, ATP should be added to the reaction to a final concentration of 0.5-1.0 mM. The 25 mM ATP Solution is included.
Inhibition and Inactivation
heat inactivated at 65°C for 10 minutes or 70°C for 5 minutes
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 is functionally tested in cloning assays and is free of detectable contaminating nuclease activities. The following QC assays are performed on each new lot:
- Endonuclease Activity (Nicking) assays
1 µg of supercoiled pUC19 plasmid DNA is incubated with 50 U of Quick Ligase in 1× Quick Ligation Buffer for4 hours at 37°C. Following incubation, the DNA is visualized on an agarose gel. There must be no visible nicking or cutting of the DNA.
- Non-Specific DNase Activity assay
The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
- Functional assay
Linearized pUC19 plasmid (leaving either blunt-end or cohesive ends) is re-ligated with 5 U of Quick Ligase. The DNA is then transformed into E. coli competent cells that are plated on ampicillin plates. The re-ligation efficiency is determined by counting transformed bacterial colonies.
Quick Ligase is shipped on dry or blue ice.
Storage and Stability
All components should be stored at -20°C. When stored under optimum conditions, the reagents are stable for a minimum of 12 months from date of purchase.