T4 DNA LIGASE: FAQs

The T4 DNA Ligase Kits from Watchmaker Genomics are ideally suited for many sensitive applications due to high-stringency enzyme manufacturing and ultra-high enzyme purity. T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the terminal 5′ phosphate and a 3′ hydroxyl group of duplex DNA, RNA, or DNA/RNA hybrids. This enzyme joins blunt and cohesive (sticky) ends, and repairs single-stranded nicks in duplex DNA,
RNA, or DNA/RNA hybrids.

One unit is defined as the amount of DNA ligase required to join 50% of 100 ng of DNA fragments with cohesive termini in 20 μL 1X DNA Ligase Buffer (final concentration 50 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, 1 mM ATP pH 7.6 @ 25°C) following a 30-minute incubation at 23°C. ATP is an essential cofactor for the ligation reaction.

Several vendors define ligase units using different unit definitions. Please reach out to support@watchmakergenomics.com to determine the concentration and volume of T4 DNA ligase to use to be a drop-in replacement for your current solution.

• Cloning of restriction enzyme-generated DNA fragments
• Cloning of PCR products
• Joining of double-stranded oligonucleotide linkers or adapters to DNA
• Site-directed mutagenesis
• Amplified fragment-length polymorphism (AFLP)
• Ligase-mediated RNA detection
• Nick repair in duplex DNA, RNA, or DNA/RNA hybrids
• Self-circularization of linear DNA

• 10 mM Tris-HCl
• 50 mM KCl
• 1 mM DTT
• 0.1 mM EDTA
• 50% glycerol pH 7.5 @ 25°C

Polyethylene glycol (PEG), has been shown to greatly increase the reaction rate and overall yield for ligation reactions. For your convenience, 5X Rapid Ligation Buffer and 2X Rapid Ligation Buffers are formulated with PEG for rapid ligation protocols. Extended ligation with PEG causes a drop off in transformation efficiency.

• 10X Ligation Buffer is formulated without PEG and is the industry standard for non-rapid ligation protocols:
  • 500 mM Tris-HCl
  • 100 mM MgCl₂
  • 50 mM DTT
  • 10 mM ATP pH 7.6 @ 25°C

• 5X Rapid Ligation Buffer is recommended to accommodate greater volumes of DNA input for rapid ligation protocols:
  • 30% PEG
  • 330 mM Tris-HCl
  • 50 mM MgCl₂
  • 5 mM DTT
  • 5 mM ATP pH 7.6 @ 25°C

• 2X Rapid Ligation Buffer is recommended for blunt ligations:
  • 15% PEG
  • 132 mM Tris-HCl
  • 20 mM MgCl₂
  • 2 mM DTT
  • 2 mM ATP pH 7.6 @ 25°C

• Use fresh ATP and include Mg2+. ATP older than one year old may lead to decreased ligation efficiency
• Ensure the sample is low in salt and has had all EDTA removed
• Keep the total DNA concentration between 1 – 10 μg/mL
• Keep the vector:insert molar ratio between 1:1 and 1:5
• When transforming your cells add between 1 – 5 μL to 50 μL competent cells
• For larger inserts reduce the insert concentration
• Ensure your competent cells are viable and run positive controls with your transformations
• Use a strain deficient in mcrA, mcrBC and mrr for inserts that may have methylated cytosines present
• For constructs larger than 10 kb, use electroporation rather than chemical transformation

Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling – including private label – designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.

Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.