CircLigase™

Get your Epicentre® Forum - hot off the press! Click here for direct link to Forum listings

Last modified: 2008-07-15 14:57:15

CircLigase™ ssDNA Ligase is a thermostable ATP-dependent ligase that catalyzes intramolecular ligation (i.e., circularization) of single-stranded DNA (ssDNA) templates having a 5'-phosphate and a 3'-hydroxyl group. In contrast to T4 DNA Ligase and Ampligase® DNA Ligase, which ligate DNA ends that are annealed adjacently to each other on a complementary DNA sequence. CircLigase™ ssDNA Ligase ligates ends of ssDNA in the absence of a complementary sequence.

The enzyme is therefore useful for making circular ssDNA molecules from linear ssDNA. Circular ssDNA molecules can be used as substrates for rolling circle replication or rolling circle transcription.

Linear ssDNA of >30 bases is circularized by CircLigase™. Under standard reaction conditions, virtually no linear concatamers or circular concatamers are produced. In addition to its activity on ssDNA, CircLigase™ also has activity in ligating a single-stranded nucleic acid having a 3'-hydroxyl ribonucleotide and a 5'-phosphorylated ribonucleotide or deoxyribonucleotide.

Figure 1. CircLigase™ ssDNA Ligase converts linear ssDNA to circular ssDNA. A 71-base ssDNA oligo was converted to a circular DNA form in a reaction containing CircLigase™ ssDNA Ligase and ATP. Lane M, DNA markers. Lane 1, 71-base ssDNA. Lane 2, circularization proceeds through an adenylated intermediate. Lane 3, the closed circular nature of the reaction product was confirmed by treating the reaction with exonuclease I, which specifically digests linear DNA.

Unit Definition:

One unit of CircLigase converts 1 picomoles of a linear 5'-phosphorylated CircLigase Standard 55-mer Oligo to exonuclease I-resistant circular form in 1 hour at 60°C under Standard Assay Conditions.

Storage Buffer:

50% glycerol containing 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 0.1 mM EDTA and 1 mM DTT.

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 25 mM solution of MnCl₂ included.

Quality Control:

CircLigase ssDNA Ligase is free of detectable phosphatase, DNA exo- and endonuclease and RNase activities.

Last modified: 2008-05-14 16:46:46

Applications

• Production of single-stranded DNA templates for rolling circle replication or rolling circle transcription experiments.
• Production of single-stranded DNA templates for RNA polymerase and RNA polymerase inhibitor assays.

Benefits

• Efficient single-stranded DNA ligase activity.
• Circularizes single-stranded DNA of >30 bases.
• Standard reaction conditions produce no detectable single-stranded DNA concatamers or concatameric DNA circles.

Last modified: 2008-05-14 16:46:46

Protocols for: CircLigase™ ssDNA Ligase

(catalogue number CL4111K / CL41115K)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46