Product Class: Other

NEBridge® Ligase Master Mix

Learn about ligase fidelity and push the limits of your Golden Gate Assembly Reactions (50+ fragments)


Catalog #M1100

Product Introduction

Offering flexibility and convenience for users, NEBridge® Ligase Master Mix performs high efficiency and high-fidelity Golden Gate Assembly with a broad assortment of NEB Type IIS restriction enzymes.

  • Optimized for efficient and accurate Golden Gate Assembly
  • Convenient 3X Master Mix format
  • Use with NEB Type IIS restriction enzymes
  • Use for seamless cloning – no scar remains following assembly
  • Ideal for ordered assembly of multiple fragments (2-25+) in a single reaction
  • Can also be used for cloning of single inserts and library construction
  • Free tool available at GoldenGate.neb.com
  • Try our free Ligase Fidelity Tools ( for the design of high-fidelity Golden Gate Assemblies)

 

 

Product Information

Description

NEBridge® Ligase Master Mix is a 3X master mix for Golden Gate Assembly. Designed for use with NEB Type IIS restriction enzymes, this master mix contains T4 DNA Ligase in an optimized reaction buffer with a proprietary ligation enhancer. Users need only choose their preferred NEB Type IIS restriction enzyme and add DNA substrates to be assembled. Low complexity single fragment insertions, as well as moderate complexity (3–6 fragment) and high complexity (7–25+ fragment) assemblies, are all supported with this optimized reagent and accompanying protocols.

25-fragment Golden Gate Assembly of lac Cassette Using NEBridge Ligase Master Mix and Conventional T4 DNA Ligase Buffer



A. Schematic of 25- fragment lac cassette assembly with NEBridge Ligase Master Mix 

B: 30 µl reactions containing 24 inserts and pGGAselect plasmid were set up using BbsI-HF (NEB #R3539M).  Left plate shows results with 10 µl NEBridge Ligase Master Mix. Right plate shows results with 3 µl of 10X T4 DNA Ligase Buffer (NEB #B0202) and 1 µl of T4 DNA Ligase (NEB #M0202T/M).  1 µl of BbsI-HF  was used for both reactions, as well as 0.05 pmol of each 24 insert DNAs and pGGAselect. Reactions proceeded for 60 cycles at 37°C for 5 min and 16°C for 5 min, with an end soak at 60°C for 5 min. 2 µl of each reaction was transformed in to 50 µl of T7 Express Competent E.coli  (NEB #C2566). 50 µl of outgrowth was plated onto LB plates (supplemented with 1 mg/ml dextrose, 1 mg/ml MgCl2, 30 µg/ml chloramphenicol, 200 µM IPTG and 80 µg/ml X-gal) and incubated at 37°C for 18 hrs, and then stored at 4°C for 4 hrs before scoring colony color phenotype. 

C:  The total transformants and percentage of correct assemblies (blue colonies) were reported as the average result of three replicates with the standard deviation from the mean. The reaction with NEBridge Ligase Master Mix generated 2.2 ±0.2X106 correctly assembled blue colonies per µg vector DNA with 94.3 ±1% fidelity, while the reaction with T4 DNA Ligase Buffer generated 8.3 ±2.1X105 correctly assembled blue colonies per µg vector DNA with 69.8 ± 10.7% fidelity. 


Reference

V. Potapov et al., Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synth Biol 7, 2665–2674 (2018).

 
This product is related to the following categories:
DNA Ligases,
DNA Assembly, Cloning and Mutagenesis Kits,
This product can be used in the following applications:
High-throughput cloning and automation solutions,
NEBridge® Golden Gate Assembly

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

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

Properties & Usage

Materials Required but not Supplied

  • User-defined DNA fragments
  • User-choice of NEB Type IIS restriction enzymes
  • Competent cells
  • Other materials for transformation

Features

  • Optimized for convenient, efficient and accurate Golden Gate Assembly
  • Use with NEB Type IIS restriction enzymes
  • Use for seamless cloning – no scar remains following assembly
  • Ideal for ordered assembly of multiple fragments (2-25+) in a single reaction
  • Can also be used for cloning of single inserts and library preparations
 

Protocols, Manuals & Usage

Protocols

  1. Golden Gate Assembly Protocol for NEBridge® Ligase Master Mix (NEB #M1100)
  2. Golden Gate Assembly Protocol using PaqCI® (NEB #R0745) and NEBridge Ligase Master Mix (NEB #M1100)

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Can Type IIS restriction enzymes from other companies be used with NEBridge Ligase Master Mix?
  2. Can I assemble PCR amplicons instead of pre-cloned inserts using Golden Gate Assembly protocols and products?
  3. How does NEBridge Ligase Master Mix perform in Golden Gate Assembly? How does it compare to “home brew” protocols?
  4. I observed high backgrounds with empty vector when using NEBridge Ligase Master Mix. What should I do?
  5. My NEBridge Ligase Master Mix has frozen in my freezer. Is this a problem?
  6. Can NEBridge Ligase Master Mix reactions be inactivated by heating?
  7. Can I use the NEBridge Ligase Master Mix reaction to transform electrocompetent cells?
  8. Which restriction enzymes are used in Golden Gate Assembly?
  9. What affects the efficiency of Golden Gate Assembly?
  10. How many base pairs should my amplicon inserts have flanking the Type IIS restriction site?

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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