Taq DNA Ligase

For joining or ligation of DNA fragments, particularly in DNA sequencing and cloning techniques

S_1319_2_LS_OEM_Taq_DNA_Ligase_20000_U
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Taq DNA Ligase (20,000 U)

Cat. No. / ID:  L6060L

Taq DNA Ligase (40,000 U/mL) and 10X Taq DNA Ligase Buffer
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The Taq DNA Ligase (20,000 U) is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM

Features

  • Thermostable DNA Ligase
  • Seals nicks
  • Discriminates against mismatch ligation

 

Product Details

Taq DNA Ligase catalyzes the formation of a phosphodiester bond in duplex DNA containing adjacent 5'-phosphoryl and 3'-hydroxyl termini, using NAD+ as a cofactor (1).

 

Supplied in:
10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Tween-20, 50% glycerol (pH 7.5 at 25°C)

Supplied with: 
10X Taq DNA Ligase Buffer (B6060): 200 mM Tris-HCl, 250 mM KCl, 100 mM MgCl2, 5 mM NAD+, 0.1% Triton X-100 (pH 7.6 at 25°C)

 

Performance

  • Storage temperature: –25°C to –15°C
  • Molecular weight: 76.9 kDa

 

Test Amount tested Specification
Purity n/a >99%
Specific activity n/a 400,000 U/mg
Single-stranded exonuclease 400 U <5.0 % released
Double-stranded exonuclease 400 U <1.0 % released
Double-stranded endonuclease 400 U No conversion
E. coli DNA contamination 400 U <10 copies

 

Principle

The enzyme is produced by a recombinant E. coli strain carrying the cloned Taq DNA Ligase gene.

One unit is defined as the amount of Taq DNA Ligase required to join 50% of 1 µg of the 12-base cohesive ends of Lambda DNA cut with SmaI and SalI in 50 µL 1X Taq DNA Ligase Buffer following a 10-minute incubation at 45°C.

 

Procedure

Usage Instructions

 

Nick ligation in double-stranded DNA

  1. Set up the following reaction mixture in a total volume of 50 µL:

    Components Final Concentration Volume
    Nuclease-free water N/A X µL
    10X Taq DNA Ligase Buffer (B6060) 1X 5 µL
    DNA up to 1µg X µL
    Taq DNA Ligase (L6060L) 80 U 2 µL
      Total Volume = 50 µL
  2. Incubate at 45°C for 10 minutes.

 

Quality Control

 

Unit activity is measured using a 2-fold serial dilution method.  Dilutions of the enzyme batch were made in 1X Taq DNA Ligase Reaction Buffer and added to 50 µL reactions containing λ Hind III digested DNA and 1X Taq DNA Ligase Reaction Buffer. Reactions are incubated for 10 minutes at 45°C, stopped, and analyzed on a 0.8% agarose gel stained with ethidium bromide.

Protein concentration (OD280) is determined by OD280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution 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.

 

Applications

This product is available for molecular biology applications such as:

  • High-temperature ligation
  • Ligase chain reaction
  • Ligase detection reaction

 

References
  1. Takahashi, M. et al. (1984). J. Biol. Chem. 259, 10041-10047.

 

Resources

Protocol Files (1)
Taq DNA Ligase Manual
PDF (216KB)
Safety Data Sheets (1)
Certificates of Analysis (1)