Product Class: Other

T7 DNA Ligase
NEBU cloned at NEB recombinant 25 No

Product Introduction

T7 DNA Ligase will ligate these substrates:

dsDNA




 

Nicked DNA/RNA


T7 DNA Ligase is an ATP-dependent dsDNA ligase from bacteriophage T7.

Catalog # Size Concentration
M0318S 100000.0 units 3000000 units/ml
M0318L 750000.0 units 3000000 units/ml

Product Information

Description

T7 DNA Ligase is an ATP-dependent ds DNA ligase from bacteriophage T7. It will catalyze the formation of a phosphodiester bond between adjacent 5´ phosphate and 3´ hydroxyl groups of duplex DNA. Cohesive end ligation and nick sealing can be efficiently catalyzed by T7 DNA Ligase (1,2). However, unlike T4 and T3 DNA Ligases, blunt end ligation is not efficiently catalyzed by T7 DNA Ligase. Addition of high concentrations of PEG 6000 [≥ 20% (w/v)] to the reaction can force T7 DNA Ligase to have measurable activity. However, under typical reaction conditions blunt-end DNA ligation does not occur in the presence of T7 DNA Ligase, making it a good choice for applications in which blunt and cohesive ends of DNA are present but only the cohesive ends are to be joined.

M0318
T7 DNA Ligase does not ligate blunt-end DNA fragments. Ligation reactions containing blunt fragments (ΦX174 DNA-HaeIII Digest (NEB #N3026 ) and sticky-end fragments (λ DNA-HindIII Digest (NEB #N3012 ) were set up with 200 ng of each substrate and 1 μl of each ligase, and incubated for 30 minutes at 25°C in their corresponding reaction buffers. Reactions were immediately stopped with 6X loading dye and resolved by electrophoresis on a 1% agarose gel and stained with ethidium bromide.

Product Source

An E. coli strain containing a recombinant gene encoding T7 DNA Ligase.
This product is related to the following categories:
DNA Ligases Products
This product can be used in the following applications:
Cloning Ligation

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to give 50% ligation of 100 ng HindIII fragments of λ DNA in a total reaction volume of 20 μl in 30 minutes at 25°C in 1X StickTogether DNA Ligase Buffer.

Reaction Conditions

1X StickTogether™ DNA Ligase Buffer
Incubate at 25°C

1X StickTogether™ DNA Ligase Buffer
66 mM Tris-HCl
10 mM MgCl2
1 mM ATP
1 mM DTT
7.5% Polyethylene glycol (PEG 6000)
(pH 7.6 @ 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

No

Application Features

  • Cloning of DNA fragments generated by restriction enzyme digestion
  • Adding linkers or adapters to dsDNA
  • Circularization of linear DNA
  • Nick-sealing in dsDNA
  • Site-directed mutagenesis

Product Notes

  1. ATP is an essential cofactor for the reaction.
  2. Dilution of enzyme for long-term storage at -20°C should be performed with the storage buffer containing 50% glycerol. Diluent A (NEB #B8001) can also be used for those applications in which BSA, present in Diluent A, will not interfere.
  3. T7 DNA Ligase is also active in buffers without PEG 6000, such as our T4 DNA Ligase Buffer and NEBuffer r1.1–r4.1, for applications in which PEG 6000 is detrimental. Please remember to supplement the reaction with 1 mM ATP (final concentration). Using these buffers, the activity of T7 DNA Ligase is reduced approximately 10-fold.
  4. Heating a reaction containing T7 DNA Ligase at 65°C for 10 minutes will inactivate the enzyme. However, the reaction needs to be performed in a buffer without PEG. Do not heat inactivate if there is PEG in the reaction buffer, as transformation will be inhibited.
  5. Standard vector + insert ligations in 10–20 μl reaction volumes are usually performed for 30 minutes at 25°C.

References

  1. Doherty, A.J. et al. (1996). J. Biol. Chem. 271, 11083-11089.
  2. Subramanya, H.S. et al. (1996). Cell. 85, 607-615.

Protocols, Manuals & Usage

Protocols

  1. Ligation Protocol with T7 DNA Ligase (M0318)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. T7 DNA Ligase is described as an enzyme that only ligates DNA fragments with cohesive ends. What length of cohesive end can T7 DNA Ligase ligate?
  2. Can T7 DNA Ligase be used with a buffer that does not contain PEG?
  3. Can T7 DNA Ligase be heat inactivated?
  4. What are some potential problems with the ligation reaction using T7 DNA Ligase that can lead to transformation failure?
  5. What are some other problems that should be considered when trouble shooting a transformation problem?
  6. What problems can be encountered in the restriction digest that can cause ligation using T7 DNA Ligase or subsequent transformation to fail?
  7. How much DNA should be used in a ligation using T7 DNA Ligase?
  8. Will T7 DNA Ligase work in rCutSmart buffer?

Troubleshooting

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 busdev@neb.com.

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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