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NEBridge® Ligase Master Mix

Catalog # Concentration Size List Price Quantity Your Price
M1100L 3 X 250 reactions $539.00
$485.10
M1100S 3 X 50 reactions $135.00
$121.50
Catalog # Size List Price Your Price
M1100L 250 reactions $539.00
$485.10
M1100S 50 reactions $135.00
$121.50
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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)

 

 
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    Golden Gate Assembly Workflow
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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).

 
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  NEBridge® Ligase Master Mix M1100SVIAL -20 1 x 0.25 ml 3 X
  NEBridge® Ligase Master Mix M1100LVIAL -20 1 x 1.25 ml 3 X
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
 
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