T4 DNA Ligase

Description

Product Description

ZELLX® T4 DNA Ligase is a high-efficiency, ATP-dependent DNA ligase derived from bacteriophage T4 and widely recognized as a trusted tool in molecular biology workflows. The enzyme catalyzes the formation of phosphodiester bonds between adjacent 5′-phosphate and 3′-hydroxyl termini, enabling reliable ligation of DNA fragments.

The enzyme exhibits high ligation efficiency, effectively joining both cohesive (sticky) ends and blunt-ended DNA fragments. This versatility makes ZELLX® T4 DNA Ligase suitable for a broad range of applications, including molecular cloning, recombinant DNA construction, adaptor and linker ligation, and next-generation sequencing (NGS) library preparation.

ZELLX® T4 DNA Ligase is supplied in a user-friendly, ready-to-use format at 5 U Weiss/µL, allowing direct application without dilution and simplifying experimental setup. Manufactured under controlled production and quality assurance conditions, each lot is validated to ensure consistent activity and reliable performance, supporting reproducible results across experiments.

Purified to high homogeneity and free of detectable DNase, RNase, and nonspecific endonuclease contamination, ZELLX® T4 DNA Ligase delivers dependable ligation efficiency for both routine and advanced molecular biology workflows in research and biotechnology laboratories.

Product Specifications:

Catalog Number: ZXB-06-205

Activity Definition:
One Weiss unit catalyzes the conversion of 1 nmol [³²P]PPi to a Norit-adsorbable form in 20 min at 37 °C and corresponds to ~200 cohesive-end ligation units.

T4 DNA Ligase Protocol

Preparation (Before Use)

1. Aliquot reagents
   It is strongly recommended to prepare aliquots of T4 DNA Ligase and 10× T4 DNA Ligation Buffer to avoid repeated freeze–thaw cycles and potential nuclease contamination.
2. Brief centrifugation
   Spin down the T4 DNA Ligase briefly before use to collect contents at the bottom of the tube.
3. Handling of 10× Ligation Buffer
   The 10× T4 DNA Ligation Buffer contains ATP, which is sensitive to temperature fluctuations.

1. • A white precipitate may occasionally be observed; this does not affect ligation performance, and the buffer can be used as is.
   • Do not heat the buffer to dissolve the precipitate, as heating may degrade ATP.

DNA Ligation Reaction:

Reaction Setup (20 µL total volume)

Prepare the following mixture in a nuclease-free tube:

• 10–100 ng linearized DNA vector (non-compatible cohesive ends or blunt-ended vectors that cannot self-ligate)
• Insert DNA: Amount corresponding to a vector: insert molar ratio of 1:1 to 1:5
• 2 µL 10× T4 DNA Ligation Buffer
• 1 µL T4 DNA Ligase (5 U/µL)
• Optional (blunt-end ligation only): 2 µL 50% PEG 4000 (not supplied)
• Nuclease-free water to 20 µL

Mix gently and briefly centrifuge.

Ligation Incubation:

1. Incubate the reaction at 22 °C for 30–60 minutes.

• Extending the incubation to 60 minutes may increase colony numbers.
• Incubation times longer than 60 minutes typically provide no additional benefit.

Optional Enzyme Inactivation

1. Heat inactivation (optional): Incubate at 70 °C for 5 minutes.

⚠️ Important:

• Do NOT heat-inactivate ligation reactions containing PEG 4000, as this significantly reduces transformation efficiency.
• When PEG is present, either:

• Use the ligation mixture directly for transformation, or
• Perform chemical inactivation by adding NaCl or KCl to a final concentration >200 mM.

Transformation:

1. Use 5–10 µL of the ligation mixture to transform 50 µL of chemically competent cells.

Important Guidelines & Tips:

• Do not exceed the recommended amount of T4 DNA Ligase in the reaction.
• For optimal transformation efficiency, the ligation mixture should not exceed 10–20% of the competent cell volume.
• DNA fragments excised from agarose gels should be handled carefully:

• UV exposure at 254–312 nm for as little as 2 minutes can reduce ligation efficiency by up to 10,000-fold due to pyrimidine dimer formation.
• Use long-wavelength UV (≈360 nm) and minimize exposure time during gel excision.

• Polyethylene glycol (PEG) significantly enhances blunt-end ligation efficiency and is strongly recommended for blunt-ended DNA.

Important Note for Electroporation:

If the ligation reaction mixture is intended for electroporation, a DNA purification step is required prior to electroporation. Salts present in the ligation buffer can cause arcing of E. coli cells, leading to failed transformations.

In this case:

Do not use heat inactivation.

Instead, purify the ligation mixture using one of the following methods:

• Spin column–based DNA purification,
• or Chloroform extraction

After purification, proceed directly with electroporation using standard protocols.