Enzymes for Molecular Biology: Enzymes for Molecular Biology

CircLigase™ II ssDNA Ligase* is a thermostable enzyme 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 II 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 >15 bases, including cDNA, is circularized by CircLigase II enzyme. Under standard reaction conditions, virtually no linear concatamers or circular concatamers are produced. In addition to its activity on ssDNA, CircLigase II enzyme also has activity in ligating a single-stranded nucleic acid having a 3"-hydroxyl ribonucleotide and a 5"-phosphorylated ribonucleotide or deoxyribonucleotide.

CircLigase II ssDNA Ligase is an improved version of our original CircLigase enzyme. CircLigase II has greater activity and a new Reaction Buffer for improved ligation efficiency.

*Covered by intellectual property rights licensed to EPICENTRE.

Unit Definition

One unit of CircLigase II enzyme converts 1 pmol of a linear 5"-phosphorylated CircLigase II 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

0.33 M Tris-Acetate (pH 7.8), 0.66 M potassium chloride and 5 mm DTT.

Quality Control

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

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Protocols for: CircLigase™ II ssDNA Ligase

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Lucigen"s product page, where the latest protocol is available.

Please note this will open a new page or window on your computer.

CircLigase™ II Protocol

(catalogue number CL9021K / CL9025K)

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

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References

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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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.

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