T4 DNA Ligase
T4 DNA ligase is isolated from a recombinant E. coli strain and is suitable for ligation of sticky- and blunt ended DNA fragments.
10 Weiss units/Ál
One unit (Weiss unit) is the amount of enzyme that catalyses the conversation of one nanomol 32P-ATP in 20 minutes at 37░C. 1 ligation unit = 0,015 Weiss unit.
20 mM Tris-HCI buffer pH 7.5; 100 mM NaCI; 0.1 mM EDTA; 2 mM DTT; 0.1 mg/ml BSA; 50% glycerol (v/v)
Store T4 DNA ligase below 0║C, preferably at -20║C, in a constant temperature freezer.
10X REACTION BUFER
660 mM Tris-HCI pH 7.5; 50 mM MgCI2; 10 mM DTE; 10 mM ATP
+ extra 1 ml 50% PEG4000 solution on request
optimal ligation occurs at 15║C
efficiency of T4 DNA LIGASE
PEG (Polyehtylenglycol) greatly increases the rate of ligation of blunt-ended DNA (1). 5% (w/v) is the suggested concentration of PEG4000 in the reaction mixture.
1. Pheiffer, B.H., Zimmermann, S.B., Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA Ligase in polymer solutions, Nucleic Acids Res., 11, 7853-7871, 1983