T4 DNA LIGASE: FAQs

PRODUCTS
What is T4 DNA Ligase?

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.

How is one unit of T4 DNA Ligase defined?

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.

How many units of T4 DNA ligase should I use to be a drop-in replacement for my current solution?

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.

What are applications for T4 DNA Ligase?
  • Cloning of restriction enzyme-generated DNA fragments
  • Cloning of PCR productsJoining 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
What is the storage buffer for T4 DNA Ligase?
  • 10 mM Tris-HCl
  • 50 mM KCl
  • 1 mM DTT
  • 0.1 mM EDTA
  • 50% glycerol pH 7.5 @ 25°C
What is the recommended reaction buffer for T4 DNA Ligase?

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
What are the best practices when performing ligation using T4 DNA Ligase?
  • 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
How long should I incubate my ligation reaction?
Both incubation time and temperature play a role in ligation efficiency and optimal conditions vary by application. Typically, a ligation reaction (blunt or cohesive ends) using T4 DNA Ligase involves incubation at 20°C. Incubation times vary by application but commonly range from 5 – 30 minutes.
What is the best practice for thawing T4 DNA ligase buffer?
At room temperature on a bench or in the palm of your hand. Thawing it at 37°C can cause the breakdown of ATP.
How can T4 DNA ligase be inactivated?
Heat inactivation may improve electrotransformation efficiency. T4 DNA Ligase can be inactivated by heating at 65°C for 10 minutes.
Can I use T4 DNA ligase to ligate adapters?
Yes, T4 DNA ligase can be used to ligate adapters appropriate for sequencing on most sequencing platforms.
What is the extinction coefficient for T4 DNA Ligase?
The extinction coefficient for T4 DNA Ligase is 0.9775 (mL/(mg*cm). If measuring protein concentration (A280), we recommend you use the protein’s extinction coefficient to ensure an accurate reading.
What are the recommended reaction setup conditions?
Please reference our technical guide for instructions for use.
Do you offer custom formats?

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.

Are you ISO 13485-certified?

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.