Properties & Usage
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.