|T7 DNA Ligase
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.
Source of Protein
A recombinant E. coli strain carrying the T7 DNA Ligase gene.
20 mM Tris-HCl
50 mM NaCl
1 mM DTT
0.1 mM EDTA
pH 7.5 @ 25°C
B1010 (2X Rapid Ligation Buffer)
2X Rapid Ligation Buffer (B1010):
20 mM MgCl2
2 mM DTT
2 mM ATP
15% PEG 6000
pH 7.6 @ 25°C
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.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 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 1.0% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 5,000 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 1.0% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 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 5uL 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.
- In the absence of high concentration crowding agents (eg 20-30% PEG 6-8000), the specific activity of T7 DNA Ligase is reduced by 1000-fold on blunt-ended fragments (1, 2).
- T7 DNA Ligase is incapable of ligating single-stranded DNA fragments (1).
- Doherty, A. et al. J.Biol. Chem (1996) V.271, No.19, 11083-11089.
- Dayal, T. Unpublished observations.
||-25 to -15°C
||30,000 U <1.0% released
||30,000 U <1.0% released
||30,000 U = No conversion
|E.coli DNA Contamination
||30,000 U <10 copies
* For a detailed summary of assay conditions and data, refer to the
Quality Controls Analysis section below
Quality Control Analysis:
Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X Rapid Ligation Buffer and added to 20 µL reactions containing double stranded DNA fragments and 1X Rapid Ligation Buffer. Reactions were incubated 30 minutes at ~23°C (room temp), plunged on ice, and analyzed on a 1% agarose gel stained with ethidium bromide.
Protein Concentration (OD280) Measurement
A 2.0 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 67,850 and molecular weight of 41,132 Daltons.
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.