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Specifications

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids, joins DNA fragments with either cohesive or blunt termini (3,4). The T4 DNA Ligase requires ATP as cofactor.
Catalog #	D3931-01
Source	E. coli cells carrying a cloned gene 30 from bacteriophage T4.
Unit Definition	One Weiss unit of the enzyme catalyzes the conversion of 1nmol of [32PPi] into Norit-adsorbable form in 20 min. at 37°C (1). One Weiss unit is equivalent to approximately 200 cohesive end ligation units (CEU)*.
	Enzyme activity is assayed in the following mixture: 66mM Tris-HCl, pH 7.6, 6,6mM MgCl2, 0.066mM ATP, 10mM DTT, 3.3uM [32PPi].
	*One CEU is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 min. at 16°C.
Storage Buffer	Supplied in: 20mM Tris-HCl pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% glycerol.
Supplied with	D3931-01A DNA Ligase T4 Reaction Buffer, 10X: 400mM Tris-HCl, 100mM MgCl2, 100mM DTT, 5mM ATP, pH 7.8
D3931-01B	PEG Solution (50% Polyethylene glycol 4000)
CAS Number	9015-85-4
Molecular Weight	~55.3kD
Inhibition and Inactivation
• T4 DNA Ligase is strongly inhibited by NaCl or KCl at concentrations higher than 200mM.
• Inactivated by heating at 65°C for 10 min or at 70°C for 5 min.
Quality Control
Endodeoxyribonuclease Assay
No conversion of covalently closed circular DNA to nicked DNA was detected after incubation of 50 units of T4 DNA Ligase with 1ug of pUC19 DNA for 4 hours at 37°C.
Ribonuclease Assay
No contaminating RNase activity was detected after incubation of 50 units of T4 DNA Ligase with 1ug of [3H]-RNA for 4 hours at 37°C.
Labeled Oligonucleotide (LO) Assay
No degradation of single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10u of T4 DNA Ligase for 4 hours at 37°C.
Blue/White Cloning Assay
pUC57 DNA/HindIII, pUC57 DNA/PstI and pUC57 DNA/SmaI digests were ligated using 10 units of T4 DNA Ligase for 1 hour at room temperature. Less than 1% of white colonies were detected after transformation of competent E. coli XL1-Blue cells with ligation mix.
Storage and Stability
May be stored at -20°C. Aliquots are stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Important Note	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.
Alternate names	EC=6.5.1.1; E. coliSupplied with Reaction Buffer