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.
Figure: 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 containing 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 overgrowth 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).
产品类别: DNA Ligases Products 应用: High-throughput cloning and automation solutions, NEBridge® Golden Gate Assembly
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