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Specifications

T4 DNA Ligase catalyzes the joining of the 3I -OH and 5I phosphate termini of duplex DNA or RNA.
Catalog #	D3931
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
CAS Number	9015-85-4
Molecular Weight	62kD
Source	E. coli
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
Concentration	1u/ul
Unit Definition
One unit of enzyme will catalyze the exchange of 1 nanomole 32PPi (pyrophosphate) into a Norit®- absorbable compound in 20 minutes at 37°C.
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
65ºC 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 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.
Alternate names	EC=6.5.1.1; E. coliSupplied with Reaction Buffer