85-1360-97　DNA Ligase T4, 5u/ul 1000u　CAS No:9015-85-4　D3931-01

特徴

• 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.
• Source:E. coli cells carrying a cloned gene 30 from bacteriophage T4.
• Unit Definition:One Weiss unit of the D3931-01 catalyzes the conversion of 1nmol of [32PPi] into Norit-adsorbable form in 20 min. at 37°C. One Weiss unit is equivalent to approximately 200 cohesive end ligation units (CEU)*1 Weiss Unit = 200cfuEnzyme 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)
• 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 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: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 after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.