What is the function of T4 DNA ligase?

Thermo Scientific 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.

Where does T4 DNA ligase come from?

T4. The DNA ligase from bacteriophage T4 (a bacteriophage that infects Escherichia coli bacteria). The T4 ligase is the most-commonly used in laboratory research. It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids.

What function is T4 DNA ligase performing what type of bond is being formed?

Catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids (1).

How do you dilute T4 DNA ligase?

To dilute T4 DNA Ligase for subsequent storage at -20 °C a stor- age buffer containing 50 % glycerol should be used, to dilute Ligase for immediate use, 1x Reaction Buffer is recommended.

How does T4 ligase buffer work?

T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes (1). Single-stranded nucleic acids are not substrates for this enzyme.

Does T4 DNA ligase need ATP?

T4 Ligase forms a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA. To perform this catalytic reaction, ligase needs ATP. In the absence of ATP, phosphodiester bond won’t form and two ends of DNA won’t hold. ATP is essential for Ligase reaction.

Does T4 DNA ligase buffer contain ATP?

Description. T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes (1).

How does DNA ligase join Okazaki fragments?

During lagging strand synthesis, DNA ligase I connects the Okazaki fragments, following replacement of the RNA primers with DNA nucleotides by DNA polymerase δ. Then, DNA ligase I binds to the PCNA, which is clamped to the nicks of the lagging strand, and catalyzes the formation of phosphodiester bonds.

How long do you incubate a T4 DNA ligase?

For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). Heat inactivate at 65°C for 10 minutes.

How is a T4 DNA ligase used in DNA?

T4 DNA Ligase T4 DNA Ligase is a ligation enzyme that can be used to join DNA fragments by catalyzing the formation of phosphodiester bonds between juxtaposed 5′ phosphate and 3′ hydroxyl termini in double-stranded DNA using ATP as a coenzyme.

How much does a T4 DNA ligase cost?

T4 DNA Ligase. 125,000 Units. $206.00. T4 DNA Ligase catalyzes the formation of phosphodiester bonds between double-stranded DNA fragments with 3′-OH and 5′-phosphate ends, in the presence of ATP. T4 DNA Ligase is supplied in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mm DTT, 0.1 mM EDTA and 50% glycerol.

Can You ligate T4 DNA at room temperature?

Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP. Room Temperature Ligation. For convenience, ligations may be done at room temperature (20-25°C).