Instant Sticky-end Ligase Master Mix

Description

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.

Reaction Volume Definition

1X Instant Sticky-end Ligase Master Mix with DNA substrates in a 10 μl reaction volume. A 10 μl reaction contains 1800 cohesive end units of T4 DNA Ligase.

Advantages and Features

Applications

  • Vector construction
  • Fragment assembly
  • Library construction

Properties and Usage

Storage Temperature

-20°C

Heat Inactivation

No

FAQs

  1. Do I need to transform the Instant Sticky-end Ligase Master Mix reactions immediately?
  2. Can the ligation reaction produced by the Instant Sticky-end Ligase Master Mix be directly used to transform electrocompetent cells?
  3. The recommended volume for an Instant Sticky-end Ligase Master Mix reaction is 10ul. I like to set-up my ligation reactions in a 20 ul volume. Can I scale-up the reaction?
  4. I routinely use more than 5 ul of my ligation reactions to transform 50 ul aliquots of competent cells. When I do this with the Instant Sticky-end Ligase Master Mix my transformation plates have very few colonies. What do you think is the problem?
  5. Can the Instant Sticky-end Ligase Master Mix be used for the ligation of blunt-end or single-base overhang fragments?
  6. My Instant Sticky-end Ligase Master Mix has frozen in my freezer. Is this a problem?
  7. The protocol for Instant Sticky-end Ligase Master Mix indicates the reaction should not be heat inactivated. How can I inactivate the ligation activity?

Protocols

  1. Ligation Protocol for Cloning with Instant Sticky-end Ligase Master Mix (M0370) 
  2. Transformation Protocol (M0370)

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

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Application Notes

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Functional Test (Ligation and Transformation):

    The product is tested for ligation and transformation efficiency using DNA containing blunt or cohesive termini. Ligation products are transformed and must exceed specifications for efficiency (transformants/µg).

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply 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.