NEB Expressions 2023 Issue II NEBridge, GGA, Golden Gate Assembly, Authenticase, error correction, gene assembly, NEBNext, FFPE, library prep, NEBuilder, DNA assembly

NEBridge Golden Gate Assembly

phi29-XT RCA Kit

Increased library yields

Improved sequencing metrics

Greater sensitivity of somatic variant calling

5-250 ng input range

NEBNext Ultrashear FFPE DNA Library Prep Kit

A Rapid 1-Day Workflow Using NEBridge® Golden Gate Assembly

Molecular biology tools enable the custom generation of proteins with complete control of sequence, purification tags, secretion signals, and other performance characteristics. While the breadth of tools allows researchers to create their desired protein, this process often involves a low-throughput and time-consuming, multiday workflow using live cells. To overcome these limitations, we have demonstrated a completely in vitro workflow that combines Golden Gate DNA Assembly, rolling circle amplification (RCA), and cell-free protein expression (CFPE) to rapidly screen the impact of multiple protein designs simultaneously.

This workflow enables researchers to generate an array of protein variants in a single day using a basic set of custom DNA vectors or insertion fragments.

Almost every tool is a collaborative project with multiple contributions from a broad range of expertise at NEB.

Sanjay Kumar, Sr. Software Engineer

Authenticase™

Authenticase (NEB #M0689) is a new, unique blend of structure-specific nucleases that can recognize and cleave outside mismatch and indel (insertion and/or deletion) regions, ranging from 1-10 base pairs on double-stranded DNA. The unique combination of enzymes in Authenticase gives it superior recognition capabilities. Authenticase identifies seven of the eight possible single-base mismatch combinations: C/C, T/C, A/C,T/G, G/G, T/T and A/A. The only single-base mismatch it does not recognize is A/G.

Archiving of clinical materials as Formalin-Fixed, Paraffin-Embedded (FFPE) samples is a common practice. However, DNA extracted from FFPE samples poses many challenges for library preparation, including low input amounts and highly variable damage from fixation, storage, and extraction methods. As a result, it can be challenging to construct high quality libraries in sufficient quantity to achieve good sequence data at the desired depth of coverage. NEB scientists have been working on repair and fragmentation solutions that result in high-quality and high-yield DNA libraries.

NEBNext FFPE DNA Library Prep Kit

NEBNext FFPE DNA Repair v2 Module

Save time

Enjoy simple and fast seamless cloning in as little as 15 minutes.

Flexibility

Use one system for both "standard-size" cloning and larger gene assembly products, up to 12 fragments.

Compatible with downstream applications

DNA can be used immediately for transformation or as template for PCR or RCA.

NEB's commitment to the scientific community through enhanced web tools

At New England Biolabs (NEB), we are steadfast in our commitment to support scientists in their research endeavors. Part of this commitment is offering an array of software tools tailored to streamline scientific processes.

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EXPRESSIONS

A scientific update

NEB

Issue II • 2023

Accelerating DNA Construction to Protein Expression

NEBridge Golden Gate Assembly (GGA)

NEBExpress® Cell-free E. coli Protein Synthesis System

Arrow and speed clock showing one-day workflow to protein

phi-29 XT RCA Kit

NEBExpress® Cell-free E. coli Protein Synthesis System

Read Feature Article

DNA parts are assembled using NEBridge Golden Gate Assembly before acting as template for a phi29-XT RCA reaction. The amplification product can then be used in a cell-free protein synthesis reaction from which protein can be purified. The entire workflow is completed in vitro.

NEBridge Golden Gate Assembly

phi29-XT RCA Kit

Select a step to view the workflow.

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NEW PRODUCT HIGHLIGHT:

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error Correction

mismatch detection assay

During gene synthesis

To estimate genome editing efficiency

applications

Error Correction

During gene synthesis

mismatch detection assay

To estimate genome editing efficiency

workflow showing mismatch detection assay

Authenticase™

Learn how Authenticase can enhance error correction during gene synthesis in our blog post

Automation-friendly workflows

NEBNext® for FFPE DNA Library Prep

View Performance Data

Read Interview

Choose NEBuilder® HiFi for DNA assembly

NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, even those with 5´- and 3´-end mismatches. Available with and without competent E. coli, this flexible kit allows simple and fast seamless cloning utilizing a new proprietary high-fidelity polymerase. Find out why NEBuilder HiFi is the next generation of DNA assembly and cloning.

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Adaptable

Adapts easily for multiple DNA manipulations, including mismatch and ssOligo assembly.

Site-directed mutagenesis

Use to perform multi-site mutagenesis.

Increased stability

Store at -20°C, with improved stability over competition.

6 reasons to choose NEBuilder HiFi

Image of elephants in a line in the sunset

infographic showing applications of NEBuilder HiFi

Therapeutics

Xenotransplantation

Livestock & crops

Food organisms

Industrial microbes

NEB is focused on discovering unique properties of Cas nucleases. Our recent offerings include EnGen® Spy Cas9 HF1 and EnGen Seq1 Cas9, RNA-guided DNA endonucleases that catalyze site-specific cleavage of double-stranded DNA (dsDNA). Both products feature the Simian virus 40 (SV40) T antigen nuclear localization signals (NLS) at both the N- and C-termini of the protein and are ideal for the direct introduction of Cas9/sgRNA complexes, underscoring their utility in precision genetic editing.

New genome editing product highlights

EnGen Seq1 Cas9

EnGen Spy Cas9 HF1

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Adding capabilities to CRISPR-Cas-based biotechnology

Molecular diagnostics

From Streptococcus equinus 5´- NAGA -3´ PAM sequence allows targeting of additional genomic regions Ideal for direct introduction of Cas9/sgRNA complexes Compatible with the EnGen Mutation Detection Kit (NEB #E3321S) Active in in vitro reactions from 20°C to 45°C

From Streptococcus pyogenes High-fidelity variant of Spy Cas9 nuclease differing by four point mutations(N497A/R661A/Q695A/Q926A) reduces off-target cleavage Targets Cas9 to the region immediately upstream of a 5′-NGG-3′PAM Ideal for direct introduction of Cas9/sgRNA complexes Compatible with EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S) and the EnGen Mutation Detection Kit (NEB #E3321S)

Globally, we possess 8.3 billion tons of plastic.The magnitude of this plastic's impact on the world cannot be overstated. By 2050, plastics alone will contribute to 15% of the global carbon budget, hastening irreversible damage caused by climate change.

Read Article

The key to advancing current recycling techniques lies in embracing enzymatic recycling. Samsara Eco has developed enzymes that depolymerize plastics into their core monomers.

by Aiden Beauglehole, Australian National University

Read Blog Post

• • • • •

EnGen Spy Cas9 HF1

EnGen Seq1 Cas9

Select a step to view the workflow.

Select a card to reveal the reason to choose NEBuilder HiFi.

23M

How many tons of plastic contaminate our aquatic ecosystems annually?

Homescreen of NEBridge Golden Gate Assembly web tool

Homescreen of NEBridge Ligase Fidelity web tools

Homescreen of EnGen sgRNA Designer web tool

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In this article, Senior Software Engineer Sanjay Kumar answers questions about the collaborative nature of tool development at NEB, the testing and validation processes, and their impact on advancing science.