CircLigase II ssDNA Ligase
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CircLigase II ssDNA Ligase is provided at 100 U/μl.
Contents: CircLigase II ssDNA Ligase, CircLigase II 10X Reaction Buffer, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Betaine, Sterile Water.
CircLigase II ssDNA Ligase
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- Intramolecular ligation of single-stranded DNA and RNA ends containing 5'-phosphates and 3'-hydroxyl groups
- ATP-independent, non-catalytic enzyme
- Circularises ssDNA 15 bases long
- Compatible with high temperature ligation reactions due to thermostability of CircLigase II enzyme
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CircLigase™ II ssDNA Ligase* is a thermostable enzyme that catalyses intramolecular ligation (i.e. circularisation) 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.
- 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.
Linear ssDNA of >15 bases, including cDNA, is circularised by CircLigase II enzyme. Under standard reaction conditions, virtually no linear concatemers or circular concatemers 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 has greater activity and a new Reaction Buffer for improved ligation efficiency.
Figure 1. CircLigase II ssDNA Ligase converts linear ssDNA
- Polidoros, A.N. et al. (2006) BioTechniques 41, 35.
- Shroff , H. et al. (2005) Nano Letters 5(7), 1509.
- Lin, C. et al. (2006) Angewandte Chemie 118, 7699.
- Korlach, J. et al. (2008) Proc. Natl. Acad. Sci. USA 105, 1176.
- McArthur, M. and Bibb, M.J. (2008) Proc. Natl. Acad. Sci. USA 105, 1020.
- Shroff , H. et al. (2008) Biophysical Journal 94, 2179.
- Kuhn, H. and Frank-Kamenetskii, M.D. (2008) Nucleic Acids Res. 36, e40.
- Murakami, T. et. al. (2008) Nucleic Acids Res. Doi:10.1093/nar/gkn1014
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 7.5), 0.66 M potassium acetate and 5 mm DTT.
Quality Control: CircLigase II ssDNA Ligase is free of detectable phosphatase, DNA exo- and endonuclease, and RNase activities.