Instant Sticky-end Ligase Master Mix is a ready-to-use 2X solution of T4 DNA ligase and a proprietary ligation enhancer in an optimized reaction buffer. Specifically formulated to rapidly ligate cohesive-end (2-4 bp) substrates and improve transformation, the mix simplifies reaction set-up and provides an optimized ratio of enzyme and buffer components. No thawing of the master mix is required as it maintains a liquid state during storage at -20°C* and no incubation time is necessary to achieve ligation efficiencies sufficient for successful sub-cloning of sticky-end substrates. Just add the master mix to DNA with compatible ends, mix and transform; thereby reducing the valuable time needed for routine ligations. Ligations for subcloning can be carried out in small volumes with low concentrations, allowing users to conserve precious DNA samples, and be used directly to transform many strains of chemically competent E. coli without dilution.
* 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 after 20 freeze/thaw cycles is close to the original mix.
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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Fragment assembly
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Library construction
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 Instant Sticky-end 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
: Ligations performed using the Instant Sticky-end Ligase Master Mix are immediately ready to be used to transform E. coli. If it is necessary to incubate the reaction prior to transformation, performance is generally good between 4-25°C. Incubation can be performed for up to one hour at the chosen temperature without negatively impacting the performance of the mix. Blunt- and TA-ends can also be ligated by this mix but require a 5-15 minute incubation time to reach our internal performance standards. The "instant" ligation criteria were met with cohesive ends of 2-4 bp in length.
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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, Instant Sticky-end 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.
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.