Thermostable ligase for intramolecular ligation (i.e. circularization) of ssDNA templates

• Circularize linear ssDNA for multiple amplification and detection applications
• Perform intramolecular ligations at high temperatures
• Generate circular DNA substrates for RCA (Rolling Circle Amplification)

CircLigase™ ssDNA Ligase and CircLigase™ II ssDNA Ligase are thermostable ATP-dependent ligases that catalyze intramolecular ligation (i.e

Applications Include:
Production of ssDNA templates for rolling-circle replication or rolling-circle transcription experiments
Production of ssDNA templates for RNA polymerase and RNA polymerase inhibitor assays

CircLigase and CircLigase II Comparison:
CircLigase is far less adenylated than CircLigase II and requires ATP in the reaction buffer. CircLigase uses a so-called "Ping-Pong, bi-bi" Michaelis-Menten reaction mechanism.
CircLigase II is nearly 100% adenylated, therefore it is not necessary to add ATP to the reaction buffer. CircLigase II works as a stoichiometric reaction, where the enzyme bonds the 5´-end of an oligo that is adenylated in the enzyme active site, and then ligates the oligo and stops. Since the reaction doesn't contain ATP, CircLigase II works in a 1:1 enzyme:oligo configuration. Once the circularization is complete, the circular ssDNA is released from the active site and the reaction stops. Note: the reaction stops when the reaction solution no longer contains free ATP.
CircLigase ligates linear ssDNA of >30 bases, while CircLigase II ligates linear ssDNA of >15 bases

Product Information:
Unit Definition: One unit of CircLigase enzyme converts 1 pmol of a linear 5´-phosphorylated CircLigase Standard 55-mer Oligo into an exonuclease I-resistant circular form in 1 hour at 60°C under standard assay conditions.
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.
Quality Control: CircLigase and CircLigase II ssDNA Ligases are free of detectable phosphatase, DNA exo- and endonuclease, and RNase activities.
CircLigase 10X Reaction Buffer: 0.5 M MOPS (pH 7.5), 0.1 M KCl, 50 mM MgCl2, and 10 mM DTT. The Reaction Buffer does not contain ATP or MnCl2 which must be added to the reaction at final concentrations of 0.05 mM ATP and 2.5 mM MnCl2. A 2.5 mM solution of ATP and a 50 mM solution of MnCl2 are included.
CircLigase II 10X Reaction Buffer: 0.33 M Tris-Acetate (pH 7.5), 0.66 M potassium acetate and 5 mm DTT.