New England Biolabs, M0659S, 5-hydroxymethyluridine DNA Kinase

Related Categories Kinases Applications DNA End Modification,, Phosphorylation (Kinase) Specification Unit Definition One unit is defined as the amount of enzyme required to protect 1 µg of Bacillus subtilis bacteriophage SP8 genomic DNA in 30 minutes at 37°C in a total reaction volume of 20 μl against subsequent cleavage by NcoI-HF restriction endonuclease. Reaction Conditions 1X T4 DNA Ligase Reaction Buffer Incubate at 37°C 1X T4 DNA Ligase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 1 mM ATP 10 mM DTT (pH 7.5 @ 25°C) Storage Buffer 300 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 10 minutes Unit Assay Conditions 1X T4 DNA Ligase Reaction Buffer, 1 µg of Bacillus subtilis bacteriophage SP8 genomic DNA in a total reaction volume of 20 μl. The reaction mix is incubated for 30 minutes at 37°C and then incubated at 80°C for 10 minutes. Confirmation of phosphorylation is determined by protection of subsequent cleavage by NcoI-HF restriction endonuclease. The reaction mix is treated with 2 μl of 10X rCutSmart Buffer and 20 units of NcoI-HF restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an agarose gel. FAQ Q: What DNA substrates has 5-hydroxymethyluridine DNA Kinase (5-HMUDK) been tested on? A: 5-HMUDK has been tested using genomic DNA from the Bacillus subtilus bacteriophage SP8. Like bacteriophages SPO1 and øe, SP8 genomic DNA contains 5-hydroxymethyldeoxyuracil (5-hmdU) fully replacing thymidine. These three 5-hmdU containing phages are available from ATCC. Q: What is the activity of 5-hydroxymethyluridine DNA Kinase (5-HMUDK) at other temperatures, buffers, etc? A: 5-HMUDK displays similar activity at 30°C and 37°C in either T4 DNA Ligase Reaction Buffer (supplied) and rCutSmart® Buffer (when supplemented with 1 mM ATP). Q: Can I purchase large amounts of an existing Enzyme for Innovation? A: Yes, if you have use for large amounts of an Enzyme for Innovation, please contact NEB at EnzymesForInnovation@neb.com. Please note that there may be limited availability and thus extended lead times required for large orders. Additional QC testing may also be necessary to meet your specific application and needs. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video.