Description
E. coli DNA ligase catalyzes the phosphodiester bond formation between an adjacent 5′ phosphate and a 3′ hydroxyl of DNA ends, requiring NAD+ and Mg2+ as cofactors. Ligation of blunt ended DNA is extremely inefficient relative to cohesive DNA end ligation and nick sealing.
Source of Protein
The gene encoding E. coli DNA Ligase expressed from a plasmid in E. coli.
Supplied in
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C
Supplied With:
B6090 (10X E.coli DNA Ligase Reaction Buffer)
10X E.coli DNA Ligase Reaction Buffer (B6090):
300 mM Tris-HCl
40 mM MgCl2
260 µM NAD
10 mM DTT
0.5 mg/mL BSA
pH 8.0 @ 25°C
Unit Definition
1 unit is defined as the amount of E.coli DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 25°C.
PRODUCT SPECIFICATION*
| Storage Temperature | -25 to -15°C |
|---|---|
| Test | Specification |
| Purity (SDS-PAGE) | >99% |
| Specific Activity | 20,000 - 28,880 U/mg |
| SS Exonuclease | 100 U < 2.0% released |
| DS Exonuclease | 100 U < 1.0% released |
| DS Endonuclease | 100 U = no conversion |
| E.coli DNA Contamination | 50 U <10 copies |
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
QUALITY CONTROL ANALYSIS
Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 20 µL reactions containing double stranded DNA fragments and 1X reaction buffer. Reactions were incubated 30 minutes at 25°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-2000 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 34,280 and molecular weight of 73,606 Daltons.
SDS-Page (Physical Purity Assessment) and Specifications
2.0 µL of 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.
CONTAMINATION TESTS
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 2.0% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate 10 µl and 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 100 Units 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 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.
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.