DNA Ligase T4, 5u/Ul (DNA) Enzyme

Molecular Biology Grade

May be stored at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawingand prior to removing the cap.

~55.3kD

9015-85-4

E. coli cells carrying a cloned gene 30 from bacteriophage T4.

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.

Supplied in 20mM Tris-HCl pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% glycerol.

MBS654009 DNA Ligase T4 Reaction Buffer, 10X: 400mM Tris-HCl, 100mM MgCl2, 100mM DTT, 5mM ATP, pH 7.8

PEG Solution (50% Polyethylene glycol 4000)

• 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.

Endodeoxyribonuclease Assay: No conversion of covalently closed circular DNA to nicked DNA was detected after incubation of 50 Weiss units of T4 DNA Ligase with 1ug of pUC19 DNA for 4hours at 37°C.

Ribonuclease Assay: No contaminating RNase activity was detected after incubation of 50 Weiss 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: The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.

Cloning; Cloning-Enzymes