Product Class: Other

Hi-T4™ DNA Ligase
NEBU 25 65 Heat


Catalog #M2622

Product Introduction

Hi-T4 DNA Ligase will ligate these substrates:

dsDNA




Nicked DNA/RNA





 

Product Information

Description

Highlights

  • Engineered thermotolerant variant of T4 DNA Ligase
  • Isolated from a recombinant source
For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page. 

A thermotolerant variant of T4 DNA Ligase, Hi-T4™ DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini in duplex DNA or RNA and is designed to function at higher temperatures than wild type T4 DNA Ligase. Hi-T4 DNA Ligase will join blunt end and cohesive end termini as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids at temperatures as high as 50ºC.

Figure 1: Hi-T4 DNA Ligase displays increased thermotolerance compared to T4 DNA Ligase. 


 
400 units of T4 DNA Ligase (NEB #M0202) or Hi-T4 DNA Ligase (NEB #M2622) were pre-incubated at 45°C in 1X T4 DNA Ligase Buffer in the absence of DNA substrate for the indicated time. Residual ligase activity was tested by adding 0.12 μM HindIII (cohesive end) fluorescently-labeled fragments and incubating at 37°C for 15 minutes. Ligation products were detected by capillary electrophoresis. 

T4 DNA Ligase steadily loses activity at 45°C, whereas Hi-T4 DNA Ligase retains full activity on a cohesive end substrate, even after 72 hours at 45°C. 


 
Figure 2: Hi-T4 DNA Ligase withstands higher temperature cycling than T4 DNA Ligase.





1,000 units of T4 DNA Ligase (NEB #M0202) or Hi-T4 DNA Ligase (NEB #M2622) in (1X) T4 DNA Ligase Buffer were cycled in the presence of DNA at 42°C (A) or 50°C (B) for 5 minutes followed by 16°C for 5 minutes in the presence of 1 μg pBR322. Following the indicated number of cycles (0, 15, 30, 45 or 60), ligase activity was tested on a fluorescently-labeled, cohesive-end substrate (HindIII overhang, 25°C for 10 minutes). Ligation products were detected by capillary electrophoresis. Remaining ligase activity (relative to 0 cycle data point) is shown.  

A. T4 DNA Ligase gradually loses activity after cycling between 16°C and 42°C (gold line, A), while Hi-T4 DNA Ligase maintains activity after 60 cycles between 16°C and 42°C (orange line, A). 

B. T4 DNA Ligase is completely inactivated after only 15 cycles between 16°C and 50°C (gold line, B), while Hi-T4 DNA Ligase maintains ~60% activity after 60 cycles between 16°C and 50°C (orange line, B).

Product Source

An E. coli strain that carries a plasmid encoding the engineered Hi-T4 DNA Ligase gene.
This product is related to the following categories:
DNA Ligases Products
This product can be used in the following applications:
BioBrick® Assembly,
Cloning Ligation,
NEBridge® Golden Gate Assembly

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to give 50% ligation of 6 µg of Lambda-HindIII DNA in 30 minutes at 25°C in a total reaction volume of 20 µl.

Reaction Conditions

1X T4 DNA Ligase Reaction Buffer
Incubate at 25°C

1X T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM DTT
(pH 7.5 @ 25°C)

Storage Buffer

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 10 minutes

Features

  • Cloning of restriction fragments
  • Joining linkers and adapters to blunt-ended DNA

Product Notes

  1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
  2. To dilute Hi-T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A, NEB #B8001S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.

  3. Ligation Temperature

    For cohesive (sticky) ends (1X T4 DNA Ligase Buffer), incubate 1 µl of Hi-T4 DNA Ligase in a 20 µl reaction between 25-50ºC for 10 minutes. For blunt ends or single base overhangs (1X StickTogether DNA Ligase Buffer), incubate 1 µl of Hi-T4 DNA Ligase in a 20 µl reaction between 25-50ºC for 10 minutes. Do not heat kill the reaction because heat treating the PEG in the StickTogether DNA Ligase Buffer will inhibit transformation.

References

  1. Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, San Diego: Academic Press.

Protocols, Manuals & Usage

Protocols

  1. Protocol for Hi-T4™ DNA Ligase (NEB #M2622)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What are some causes of ligation reaction failure that can lead to transformation failure?
  2. When should Hi-T4 DNA Ligase be the enzyme of choice?
  3. Which buffer should I use with Hi-T4 DNA Ligase?
  4. What is the activity of Hi-T4 DNA Ligase in other buffers?
  5. Can this ligase be heat inactivated?
  6. What is the difference between the unit definitions of T4 DNA Ligase (NEB #M0202) and Hi-T4 DNA Ligase (NEB #M2622)?
  7. How much DNA should be used in a ligation using this ligase?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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