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 37C. One Weiss unit is equivalent to approximately 200 cohesive end ligation units (CEU)* 1 Weiss Unit = 200cfu 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 16C. 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 65C for 10 min or at 70C 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 37C. 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 37C. 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 37C. 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 -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Molekulargewicht:
55.3
CAS Nummer:
[9015-85-4]
* Mehrwertsteuer und Versandkosten nicht enthalten. Irrtümer und Preisänderungen vorbehalten
