| <b>Unit Definition</b> One unit is defined as the amount of enzyme required to protect 0.5 µg T4gt-DNA against cleavage by MfeI restriction endonuclease. <b>Reaction Conditions</b> 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) <b>Diluent Compatibility</b> Diluent B <b>Storage Buffer</b> 20 mM KPO4 200 mM NaCl 0.25 mM DTT 0.1 mM EDTA 50% Glycerol pH 7 @ 25°C <b>Heat Inactivation</b> 65°C for 10 minutes <b>Molecular Weight</b> Theoretical | 40666 kDa <b>Unit Assay Conditions</b> 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. |
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