T3 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. In the absence of 20-30% PEG 6000, T3 DNA Ligase displays a very low efficiency for blunt-ended ligation. (1) T3 DNA Ligase displays a higher efficiency for joining A/T overhangs than C/G matched ends. (1) T3 DNA Ligase retains 95% of its activity in 1.0 M NaCl or KCl, with an optimal concentration of 300 mM(1).
Source of Protein
A recombinant E. coli strain carrying the T3 DNA Ligase gene.
20 mM Tris-HCl
300 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 T3 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 23°C.
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 60,880 and molecular weight of 39,350 Daltons.
SDS-Page (Physical Purity Assessment) and Specifications
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.
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.
|T3 DNA Ligase|
|Unit Size||900,000 U|
|SDS||Available on request|
|Storage Temperature||-25 to -15°C|
|Specific Activity||3,000,000 U/mg|
|SS Exonuclease||30,000 U <1.0% released|
|DS Exonuclease||30,000 U <1.0% released|
|DS Endonuclease||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.
T3 DNA Ligase is supplied with 2X Rapid Ligation Buffer for use in ligation reactions.
The enzyme has also been characterized in the following buffer, with a 17-fold decrease in specific activity due to the absence of the crowding agent (PEG 6000):
1X T3 DNA Ligase Buffer:
50 mM Tris-HCl
300 mM NaCl
0.5 mM ATP
1 mM DTT
pH 8.0 @ 25°C
The following information was taken from the Cai reference (cited below), which describes the characterization of the T3 DNA Ligase:
Optimal pH: 8.0
Optimal reaction temperature: 20°C
Optimal reaction time: >30 minutes (without PEG 6000)
Optimal ionic strength: 300 mM NaCl
Optimal divalent cation/concentration: 2 mM Mg++
Optimal cofactor: ATP (0.5 mM)
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.
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