New England Biolabs, M0357S, T4 Phage β-glucosyltransferase (T4-BGT)

T4 Phage β-glucosyltransferase specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA, making beta-glucosyl-5-hydroxymethylcytosine (1,2). Related Categories Hydroxymethylation Detection and Analysis,, Enzymatic Conversion for DNA Methylation Analysis,, DNA Methylation Analysis Specification Unit Definition One unit is defined as the amount of enzyme required to protect 0.5 µg T4gt-DNA against cleavage by MfeI restriction endonuclease. Reaction Conditions 1X NEBuffer™ 4 Supplement with 40 µM Uridine Diphosphate Glucose Incubate at 37°C 1X NEBuffer™ 4 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 1 mM DTT (pH 7.9 @ 25°C) Diluent Compatibility Diluent B Storage Buffer 20 mM KPO4 200 mM NaCl 0.25 mM DTT 0.1 mM EDTA 50% Glycerol pH 7 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Theoretical: 40666 kDa Unit Assay Conditions 0.5 µg T4gt-DNA, 1X NEBuffer 4 and 40 µM UDP-Glucose in a 30 μl reaction. Incubate for 1 hour at 37°C followed by 10 minutes at 65°C. The extent of protection by T4 -BGT is determined by the addition of 20 μl 1X NEBuffer 4 and 10 units of MfeI. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels. Enzyme Properties Activity in NEBuffers: NEBuffer 1 100 NEBuffer 2 50 NEBuffer 3 50 NEBuffer 4 100% Survival in a Reaction: A minimum of 0.16 unit for 16 hours is required to protect 0.5 µg T4 gt-DNA against cleavage by MfeI. FAQ Q: Will T4 Phage β-glucosyltransferase (T4-BGT) work in rCutSmart Buffer? A: T4 Phage β-glucosyltransferase (T4-BGT) will work in rCutSmart Buffer. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart.