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Cat. No.: T3DL-150k (for 1250U)

Cat. No.: T3DL-600k (for 5KU)

Cat. No.: T3DL-3m (for 3MU)

Description

T3 DNA Ligase efficiently catalyzes the ligation of cohesive and blunt-ended double-stranded DNA molecules, as well as the repair of nicks in double-stranded DNA or DNA-RNA hybrid duplexes. In the case of nick repair, the intact strand can be DNA, and the nicked strand with 5' and 3' ends as DNA-DNA, DNA-RNA, RNA-DNA, or RNA-RNA can all be repaired by T3 DNA Ligase. Therefore, T3 DNA Ligase can also be used to generate DNA-RNA and RNA-DNA fusion junctions, as well as for RNA ligation based on DNA templates to form longer RNA fragments.

T3 DNA Ligase catalyzes the formation of a phosphodiester bond between adjacent 5' phosphate and 3' hydroxyl groups in double-stranded DNA, and it exhibits higher efficiency in connecting A/T overhang ends compared to C/G ends.

Similar to T4 DNA Ligase, the addition of PEG6000 in the reaction system of T3 DNA Ligase significantly improves its efficiency in ligating blunt-ended double-stranded DNA. In the absence of PEG6000, T3 DNA Ligase has lower efficiency in ligating blunt-ended double-stranded DNA molecules. Furthermore, T3 DNA Ligase has twice the tolerance to NaCl compared to T4 DNA Ligase, maintaining 95% activity under conditions of 1.0M NaCl or KCl. Therefore, in experiments involving high ion concentrations, T3 DNA Ligase is an ideal choice.

Application

Cloning of DNA fragments cut by restriction endonucleases, cloning of PCR products, ligation of double-stranded DNA and adapters, circularization of linear double-stranded DNA, nick repair in double-stranded DNA, site-directed mutagenesis, ligation in high salt systems, nick repair in double-stranded DNA, DNA-guided DNA-RNA ligation, DNA-guided RNA ligation.

Source

Purified from recombinant E.coli strain carrying the T3 bacteriophage DNA ligase gene.

Activity Definition

One unit is defined as the amount of enzyme required to achieve 50% ligation of 100ng HindIII fragments of λ DNA in a total reaction volume of 20μl in 1 minute at 25°C in 1X T3 DNA Ligase Reaction Buffer.

Purity

Free of any other DNA ligases except T3 DNA Ligase, no endonucleases or exonucleases, no RNAase, no phosphatases.

Enzyme Storage Buffer

10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH7.4 @25°C).

2X Reaction Buffer

132mM Tris-HCl, 20mM MgCl2, 2mM DTT, 2mM ATP, 15% Polyethylene glycol (PEG6000) (pH7.6 @25°C).

Inactivation or Inhibition

Incubation at 65°C for 10 minutes in a reaction system without PEG6000.

Precautions

• ATP is an essential cofactor for the catalytic activity of T3 DNA Ligase, unlike E.coli DNA Ligase which requires NAD as a cofactor. Therefore, it is necessary to maintain a final concentration of 1mM ATP in the ligation reaction system. The provided 2X Reaction Buffer already contains ATP and PEG6000 required for ligation of sticky ends.
• T3 DNA Ligase catalyzes the ligation of double-stranded DNA or nicked double-stranded DNA with the complete chain as DNA. It cannot be directly used for the ligation of single-stranded DNA or RNA. However, it can be used for the ligation of single-stranded DNA guided by a DNA template, ligation of single-stranded RNA, or ligation between single-stranded DNA and RNA.
• The reaction system of T3 DNA Ligase contains 7.5% PEG6000. If the subsequent experimental system is not compatible with PEG6000, you can consider preparing a ligation reaction buffer without PEG6000 or using the ligation buffer system of T4 DNA Ligase, while ensuring the addition of a final concentration of 1mM ATP. However, the activity of T3 DNA Ligase in the ligation buffer system of T4 DNA Ligase will be reduced by approximately 10-fold.
• In a reaction system without PEG, T3 DNA Ligase can be heat inactivated. However, if the reaction system contains PEG6000, T3 DNA Ligase cannot be heat inactivated as it will significantly reduce subsequent transformation efficiency.
• If it is necessary to maintain a high concentration of NaCl in the ligation system, it is recommended to use a buffer solution without PEG6000.
• In a standard 20μl reaction system for vector and fragment ligation, a reaction time of 30 minutes at 25°C is typically required.
• High-quality ultrapure water is recommended for the reaction system.
• This product is intended for use by professionals in scientific research and is not intended for clinical diagnosis or treatment. It should not be used for food or drug purposes and should not be stored in residential areas.
• For your safety and health, please wear lab coat and disposable gloves during operation.

Storage

The minimum shelf life is 2 years at -20°C.

DNA Ligase Products:

• T4 DNA Ligase
• Rapid T4 DNA Ligase
• T4 DNA Ligase (Fast)
• T7 DNA Ligase
• E. coli DNA Ligase
• PBCV-1 DNA Ligase
• T3 DNA Ligase
• Taq DNA Ligase
• Pfu DNA Ligase
• High-Fidelity DNA Ligase
• High-Salt DNA Ligase

SBS Genetech is recognized as one of the global major leading industry players in Ligase Market by third-party market researchers. For more details, please visit Ligase Market - A Global and Regional Analysis Focus on Product, Source, Application, End User, and Country - Analysis and Forecast, 2022-2032