Master Mix From New England Biolabs for Easy Plasmid Generation

November 20, 2023

Robert Bosch Gesellschaft für medizinische Forschung mbH
IKP/RBCT
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

New England Biolabs Inc.

Product Name:

NEBuilder® HiFi DNA Assembly Master Mix

Catalog Number:

E2621

The NEBuilder system from New England Biolabs is a very convenient tool for building your plasmids. With specific primers created with an online tool the system usually works fine and successful generation usually depends on the number of fragments and their size and quality.

Experimental Design and Results Summary

Application

Plasmid cloning/generation

Starting Material

Plasmid backbone/PCR DNA fragments and NEBuilder-specific primers

Tips

Use this online tool for creating your primers: nebuilder.neb.com. The tool is quite intuitive as long as you are specific about the sequences of your starting material. Also tools like NEBio calculator is convenient to determine needed amounts of fragments/backbone.

Results Summary

The reaction with the NEBuilder Hifi DNA Assembly master mix only takes 1h at 50°C. Afterwards the product can be controlled by PCR or directly amplified in bacteria.Sequences are most often ligated correctly with up to 4 fragments in our experiments. Optimisation seldomly needed - usually when your sequence input was not 100 % correct.

DOI or PMID #

N/A

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Summary

The Good

Quick reaction, no other reagents needed, and works with very little material. Availability of online tool for assistance.

The Bad

Long preparation process (primer/fragment generation).

The Bottom Line

The NEBuilder system is a worthy alternative to other plasmid generation systems and works well with up to 4 fragments in our hands. Starting materials should be evaluated carefully but then the reaction usually gives satisfying results.

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