T7 DNA Ligase

Description:
T7 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl termini in duplex DNA. The enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA.

Applications:

• joining of Okazaki fragments during replication
• completing short-patch DNA synthesis occurring in DNA repair process

Source:
A recombinant E. coli strain carrying the T7 DNA Ligase gene.

Specific Activity:
3,000,000U/mg

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

2X Rapid Ligation Buffer
132mM Tris-HCI
20 mM MgCl2
2 mM dithiothreitol
2 mM ATP
15% PEG 8000
pH 7.6 @ 25°C

Unit Definition:
1 unit is defined as the amount of T7 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 23°C.

Nuclease Contamination Tests

Single-Stranded Exonuclease Activity:
A 50 µL reaction containing 20,000 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 15,000 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 pBR322 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.

E.coli 16S rDNA Contamination Test:
Replicate 5 µL samples of enzyme solution were heat denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus. Based on no template control Ct values, the detection limit of this assay is <10 copies genome/sample.

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 Rapid Ligation Buffer ([T7 DNA Ligase]f = 0.23-15 µg/µL) and added to 50 µL reactions containing 0.1 µg DNA and 1X Rapid Ligation Buffer. Reactions were incubated 30 minutes at 23ºC (room temp), plunged on ice, and analyzed on a 1% agarose gel stained with ethidium bromide. 1 unit is defined as the amount of T3 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 50 µL following a 30 minute incubation at 23°C.

Protein Concentration (OD280) Measurement:
A 3.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 68,210 and molecular weight of 41,132 Daltons. Acceptance for this assay is +/- 5% of reference sample.

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 sample. 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 Condition:
 -20°C

1. Doherty, A. et al. J.Biol. Chem (1996) V.271, No.19, 11083-11089.