Blunt/TA Ligase Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer. This master mix is specifically formulated to improve ligation and transformation of both blunt-end and single-base overhang substrates. The master mix format simplifies reaction set-up, ensures an optimized ratio of enzyme and buffer components, and yields robust, rapid ligation of all types of DNA ends using a short incubation time at room temperature. No thawing is necessary as it remains liquid during storage at -20°C.* Ligations for subcloning can be carried out in small volumes with low DNA concentrations, allowing users to conserve precious DNA samples and directly transform many strains of chemically competent E. coli without dilution.
Learn more about Joining Difficult to Ligase dsDNA Fragments and Efficient Adaptor Ligation for the Preparation of dsDNA Libraries using Blunt/TA Ligase Master Mix.
Blunt/TA Ligase Master Mix improves yields for ends that typically react slowly
Yields of final ligation product for all reaction conditions using high concentration T4 DNA Ligase (NEB #M0202), the Quick Ligation Kit (NEB #M2200) and Blunt/TA Master Mix (NEB #M0367). Nick, cohesive end and 3´ single-base overhang substrates were incubated for 15 minutes; the 5´ single-base overhang was incubated for 1 hour. * Freezers vary in their actual internal temperature. Our testing demonstrates that the master mix is liquid at -20°C. Freeze-thaw testing at -70°C has confirmed that the performance is unchanged after 20 freeze/thaw cycles.
Ligation reactions with single-base overhang vector and insert were set-up using the Blunt/TA Ligase Master Mix and incubated for different times at 25°C (A) or at different temperatures for 15 minutes (B). Two microliters of each reaction were used to transform a 50 μl aliquot of NEB 10-beta Competent E. coli (NEB #C3019). Transformants resulting from triplicate plating 50 μl of a 1:5 dilution of the outgrowth were counted and graphed. The results indicate that the Blunt/TA Ligase Master Mix works well at 25°C, and is complete in 15 minutes.
Product Source
Purified from an E. coli strain containing a recombinant gene encoding T4 DNA Ligase.
Reagents Supplied
The following reagents are supplied with this product:
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Advantages and Features
Application Features
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Vector construction
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Linker ligation
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Fragment assembly
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Library construction
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TA cloning
Properties & Usage
Related Products
Product Notes
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DNA:
Purified DNA for ligations can be dissolved in dH2O (Milli-Q® water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20-100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 10 μl, increase the volume of Blunt/TA Ligase Master Mix such that it remains 50% of the reaction. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
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Time and Temperature:
Most ligations performed using the Blunt/TA Ligase Master Mix reach an end point at 60 minutes or less when performed between 4-37°C. Incubation beyond this time provides no additional benefit. Our recommendation for a 25°C (room temperature) incubation was chosen after evaluation of performance at 4°C, 16°C, 25°C, and 37°C. Most conditions reached at least 50% performance within 15 minutes. Shorter times can also be used.
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Cells:
Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes.
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Electroporation:
While electroporation can dramatically increase transformation efficiency, Blunt/TA Ligase Master Mix is not directly compatible with transformation by electroporation. It is necessary to reduce the PEG concentration. We recommend purification of the ligated DNA by spin column.
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Biology:
Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.
References
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Quick J., Quinlan, A.R., Loman N.J. (2014). A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer. Gigascience. 3, 22. DOI: 10.1186/2047-217X-3-22 PubMedID: 25386338
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Madoui M.A., Engelen S., Cruaud C., Belser C., Bertrand L., Alberti A., Lemainque A., Wincker P., Aury J.M. (2015). Genome assembly using Nanopore-guided long and error-free DNA reads. BMC Genomics. 16, 327. DOI: 10.1186/s12864-015-1519-z PubMedID: 25927464
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Karamitros T., Magiorkinis G. (2015). A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits. Nucleic Acids Res.. 15, 43. DOI: 10.1093/nar/gkv773 PubMedID: 26240383
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Karlsson E., Lärkeryd A., Sjödin A., Forsman M., Stenberg P. (2015). Scaffolding of a bacterial genome using MinION nanopore sequencing. Sci Rep.. 5, 11996. PubMedID: 26149338
Protocols
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.)
Selection Charts
Selection Charts
Usage Guidelines
Quality Control Assays
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.
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]
Specifications & Change Notifications
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]
The following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.)
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please email us at 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.