Joining dsDNA with cohesive or blunt termini Joining synthetic linkers or adapters to blunt-ended DNA Repairing nicks in duplex DNA, RNA or DNA/RNA hybrids Ligase mediataed TNA detection Site-directed mutagenesis

D3931-01A: DNA Ligase T4 Reaction Buffer, 10X

20mM Tris HCl, pH 7.5, 0.1mM EDTA, 1mM DTT, 50mM KCl and 50% glycerol

400mM Tris HCl, 100mM MgCl2, 100mM DTT, 5mM ATP

One unit of enzyme will catalyze the exchange of 1 nanomole 32PPi (pyrophosphate) into a Norit®- absorbable compound in 20 minutes at 37°C.

SDS-PAGE Contains no detectable endo-, exodeoxyribonucleases, ribonucleases. Tested for the cohesive and blunt-ended DNA fragment joining.

66mM Tris-HCl, pH 7.6 6.6mM MgCl2 0.066mM ATP 10mM DTT, 3.3uM ( 32PP)

65ºC for 10 minutes. One Weiss unit equals ~200 cohesive- end ligaton units. One unit is defined as as the amount of enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 min at 16ºC in 20ul of the assay mixture: 50mM Tris-HCl (pH 7.5), 10mM MgCl2, 10mM DTT, 1mM ATP, 25µg/ml BSA and a 5'-DNA termini concentration of 0.12µM (300µg/ml). The ratio of Weiss unit to cohesive-end ligation unit is determined by conversion of [5'-33P]-labeled termini of HindIII fragments of lambda DNA to a phosphatase-resistant form. Polyethylene glycol (PEG) greatly increases the rate of ligation of blunt-ended DNA (9). 5% (w/v) is the suggested concentration of PEG 4000 in the reaction mixture. The T4 DNA Ligase binding to DNA may result in a band shift in agarose gels. To avoid this, prior to electrophoresis we recommend to incubate the samples with 6X Loading Dye & SDS Solution at 65°C for 10min and chill on ice. It is necessary to remove the enzyme from the ligation mixture by chloroform extraction prior to electro-transformation of bacterial cells with DNA.

E. coli

~1u/ul

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.