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Plasmids 101: Protein Expression

Posted by Alyssa Cecchetelli on Jun 7, 2018 9:17:55 AM

The central dogma in molecular biology is DNA→RNA→Protein. To synthesize a particular protein DNA must first be transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into polypeptide chains that make up the primary structure of proteins. Most proteins are then modified via an array of post-translational modifications including protein folding, formation of disulfide bridges, glycosylation and acetylation to create functional, stable proteins. Protein expression refers to the second step of this process: the synthesis of proteins from mRNA and the addition of post-translational modifications

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Topics: Plasmids 101, Plasmid How To

Plasmids 101: Walkthrough of Addgene’s Snapgene-Powered Quality Control Process

Posted by Amanda Hazen on Feb 1, 2018 10:07:31 AM

We’ve talked a lot about the quality control process at Addgene by introducing our new sequencing partner seqWell and going into detail about how we use next generation sequencing results to perform quality control on deposited plasmids. We’ve also talked about how our new Snapgene generated maps provide improved feature detection with an easy to use interface. We regularly use Snapgene for our quality control process because of its expansive feature library and useful tools. In this blog, we’ll walk you through how a scientist at Addgene uses Snapgene to confirm the sequence of a plasmid and we’ll highlight some of the new features available on our website through our Snapgene powered maps and sequence analysis tools.

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Topics: Plasmids 101, Plasmid How To, Plasmid Cloning

How to Design Your gRNA for CRISPR Genome Editing

Posted by Guest Blogger on May 3, 2017 11:00:00 AM

This Post was updated on May 3, 2017 with additional information and resources. 

This post was contributed by guest blogger, Addgene Advisory Board member, and Associate Director of the Genetic Perturbation Platform at the Broad Institute, John Doench.

CRISPR technology has made it easier than ever both to engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post will discuss differences between these approaches, as well as provide updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment in Addgene's Validated gRNA Sequence Datatable.

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Topics: Plasmid How To, Genome Engineering, Lab Tips, CRISPR

The Power Behind NGS Plasmid Validation: seqWell

Posted by Guest Blogger on Apr 19, 2017 11:25:29 AM

This post was contributed by guest blogger Joe Mellor, Founder and CEO of seqWell Inc.

Plasmids and PCR products are the bread and butter of molecular biology labs the world over. Scientists have traditionally used Sanger sequencing to validate these constructs, as the relatively low cost and quick turn-around time of Sanger sequencing have historically matched the needs of most molecular biology labs. Recent and rapid advances in technologies that permit large-scale creation and synthesis (“writing”) of longer pieces of synthetic DNA, as well as the advent of extremely fast, cheap and accurate sequencing (“reading”) of DNA, have changed our collective thinking about the feasible size and scope of projects in many labs. However, the high costs of sample preparation for high-throughput next generation (NGS) sequencing have prevented laboratories from using these methods for routine processes like plasmid validation.

At seqWell, Inc., our mission is to overcome crucial challenges in NGS by developing technologies that can help unlock the potential of modern sequencing instruments by enhancing the efficiency and simplicity of library prep. As part of our mission, we’ve been working with Addgene to develop and apply our plexWell™ Library Preparation Technology for NGS-based sequencing and confirmation of Addgene’s large and growing collection of curated plasmids from all over the world. The rest of this piece will describe plexWell™ in more detail, and how we are using this technique in our partnership with Addgene to sequence large numbers of plasmids.

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Topics: Inside Addgene, Plasmid How To

Plasmids for Endogenous Gene Tagging in Human Cells

Posted by Guest Blogger on Apr 6, 2017 9:02:59 AM

This post was contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen Institute website at alleninstitute.org.

A classic challenge in cell biology is making sure that what we observe through the microscope represents reality as accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can both interfere with a cell’s normal function and result in a ubiquitous background signal that makes it hard to visualize the precise location of the protein or structure of interest.

Endogenous gene tagging is an ideal solution because it allows for tagging and visualization of specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise location in the genome is still inefficient, particularly in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving normally can be prohibitively expensive for many labs.

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Topics: CRISPR, Plasmid How To, Techniques

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