ArcticZymes R2D LigaseTM
Novel substrate specificity
RNA to DNA ligation
+++ almost complete ligation of substrate
++ about 50% ligation of substrate
+ detectable ligation activity (<< 50%)
Figure 1: ArcticZymes R2D Ligase exhibits the unique feature of being able to ligate DNA to both the 5’- and the 3’ end of RNA in the presence of a DNA template positioning the ligatable ends of DNA and RNA. When compared to other ligases, the efficiency of this activity in ArcticZymes R2D Ligase clearly stands out as proven when using a fluorescein labelled oligo neighboring a phosphorylated oligo forming a nick. Successful ligation of the nick increases the length of the fluorophore-labelled oligo resulting in slower migration during gel separation. AZ Prototype L18 and L19 are novel ligases soon to be available as prototypes.
Source: Recombinantly expressed in E. coli.
Unit Definition: One milliunit is defined as the amount of enzyme needed to ligate 1 pmol (of 18 pmol) of a nicked DNA substrate in 20 minutes at 25 °C in a 20 µl reaction volume in a buffer consisting of 62.5 mM Tris-HCl, pH 7.5 (25°C), 5 mM MgCl2, 1 mM ATP, 10 mM DTT, 0.025 mg/ml BSA and 25 mM KCl.
Size: 52.9 k Da
Storage and Stability: The enzyme is stable at -20°C for 1 year in the supplied storage buffer.
ArcticZymes is dedicated to the quality of our products. R2D DNA Ligase is manufactured at our ISO 13485 certified facility in Norway.
dsDNA endonuclease activity
2.5 U R2D Ligase was incubated with a supercoiled plasmid (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any transformation of closed circular DNA to nicked DNA.
ssDNA endonuclease activity
2.5 U R2D Ligase was incubated with M13 ssDNA (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of ssDNA degradation.
2.5 U R2D Ligase was incubated with either 3H-dATP labelled ds or ssDNA (0.5 µg, 500 bp) for 4 hours at 37°C. Acid soluble radioactivity from labelled DNA was not significantly over blank test for either substrate.
2.5 U R2D Ligase was incubated with a 2 kb RNA transcript (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of RNA degradation.
E. coli gDNA contamination
2.5 U R2D Ligase was analysed in a probe-based qPCR assay detecting the 23S ribosomal subunit in E. coli. No E. coli gDNA could be detected (LOD: < 3 E. coli genomic copies).
Additional Data and Information
We are pleased to provide additional data relating to ArcticZymes R2D Ligase upon request.
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