T4 DNA Ligase

Description:
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the terminal  5′ phosphate and a 3′ hydroxyl groups of duplex DNA or RNA. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids⁽¹⁾.

Applications:

• Cloning of restriction fragments
• Joining linkers and adapters to blunt-ended DNA

Source:
A recombinant E. coli strain carrying the cloned T4 DNA Ligase gene.

Specific Activity:
300,000U/mg

Supplied In:
10 mM Tris-HCl
50 mM NaCl
1 mM dithiothreitol
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C

Supplied With:
10X T4 DNA Ligase Buffer

10X T4 DNA Ligase Buffer
500mM Tris-HCI
100 mM MgCl2
50 mM dithiothreitol
10 mM ATP
pH 7.6 @ 25°C

Nuclease Contamination Tests

Single-Stranded Exonuclease Activity:
A 50 µl reaction containing 15,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.1% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity:
A 50 µl reaction containing 10,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.1% release of TCA-soluble counts.

Endonuclease Activity:
A 50 µL reaction containing 1 µg of pENZuC DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

Real-Time PCR DNA Contamination Test:
Replicate 5 µL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values and standard curve data, the detection limit of this assay is <10 copies genome/sample.

Molecular Weight:
55.3 kDa

Unit Definition:
One unit of the enzyme catalyzes the conversion of 1 nanomole of [32PP] into Norit-adsorbable form in 20 min at 37ºC (Weiss unit). One Weiss unit is equivalent to approximately 200 cohesive-enc ligation units.
One cohesive-end ligation unit is defined as the amount of enzyme required to give 50% ligation of Hidlll fragments of lambda DNA IN 30MIN at 16ºC in 20μl of the assay mixture: 50mM Tris-HCL (pH 7.5), 10mM MgCl2, 10mM DTT, 1mM ATP, 25μg/ml BSA and a 5’-DNA termini concentration of 0.12μM (300μg/ml).

Quality Control Analysis

Unit Characterization Assay:
Unit activity was measured using a 2-fold serial dilution method.  Dilutions of enzyme batch were made in 1X T4 DNA Ligase Reaction Buffer ([T4 DNA Ligase]f = 0.31-20 µg/µL) and added to 50 µL reactions containing 0.1 µg DNA and 1X T4 DNA Ligase Reaction Buffer. Reactions are incubated for 30 minutes at 23°C, stopped, and analyzed on a 1% agarose gel stained with ethidium bromide.

SDS-Page (Physical Purity Assessment):
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the same enzyme species.   Following electrophoresis, the gel was stained and the samples compared to determine physical purity.  The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

Recommended Storage Conditions:
 -20°C

References:
1. Engler, M.J. and Richardson, C.C. (1982) P.D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego: Academic Press.