T4 DNA Ligase catalyzes the joining of the 3I -OH and 5I phosphate termini of duplex DNA or RNA. Applications: 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 Supplied With: D3931-01A: DNA Ligase T4 Reaction Buffer, 10X Storage Buffer: 20mM Tris HCl, pH 7.5, 0.1mM EDTA, 1mM DTT, 50mM KCl and 50% glycerol Reaction Buffer (10X): 400mM Tris HCl, 100mM MgCl2, 100mM DTT, 5mM ATP Unit Definition: One unit of enzyme will catalyze the exchange of 1 nanomole 32PPi (pyrophosphate) into a Norit- absorbable compound in 20 minutes at 37C. Quality Control: SDS-PAGE Contains no detectable endo-, exodeoxyribonucleases, ribonucleases. Tested for the cohesive and blunt-ended DNA fragment joining. Activity Assay: 66mM Tris-HCl, pH 7.6 6.6mM MgCl2 0.066mM ATP 10mM DTT, 3.3uM ( 32PP) Inactivation: 65C for 10 minutes. Note: 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 16C 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 65C 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.
Molecular Weight:
62
CAS Number:
[9015-85-4]
* VAT and and shipping costs not included. Errors and price changes excepted
