Enzymes for Molecular Biology: Enzymes for Molecular Biology

Applications

• 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™ ssDNA Ligase* is a thermostable ATP-dependent ligase that catalyzes intramolecular ligation (i.e. circularization) of 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 adjacent 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 enzyme. Under standard reaction conditions, virtually no linear concatamers or circular concatamers are produced. In addition to its activity on ssDNA, CircLigase enzyme also has activity in ligating a single-stranded nucleic acid having a 3´-hydroxyl ribonucleotide and a 5´-phosphorylated ribonucleotide or deoxyribonucleotide.

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protocol can be be downloaded from the manufacturers website here

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Product Citations

1. Polidoros, A.N. et al. (2006) BioTechniques 41, 35.
2. Shroff , H. et al. (2005) Nano Letters 5(7), 1509.
3. Lin, C. et al. (2006) Angewandte Chemie 118, 7699.
4. Korlach, J. et al. (2008) Proc. Natl. Acad. Sci. USA 105, 1176.
5. McArthur, M. and Bibb, M.J. (2008) Proc. Natl. Acad. Sci. USA 105, 1020.
6. Shroff , H. et al. (2008) Biophysical Journal 94, 2179.
7. Kuhn, H. and Frank-Kamenetskii, M.D. (2008) Nucleic Acids Res. 36, e40.
8. Murakami, T. et. al. (2008) Nucleic Acids Res. Doi:10.1093/nar/gkn1014

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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.

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.

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

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