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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: Plasmid How To, Inside Addgene

Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

Posted by Beth Kenkel on Apr 13, 2017 10:30:00 AM

Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark Prescott. This system was developed for monitoring and analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a more neutral pH compartment, ­­like the cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella improves upon them.

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Topics: Fluorescent Proteins

Plasmids 101: Aptamer Fluorophores

Posted by Eric J. Perkins on Apr 11, 2017 10:30:00 AM

What is an Aptamer?

Nearly 30 years ago, two independent groups, led by Jack Szostak and Larry Gold, developed methods for selecting and amplifying RNA sequences that could bind very specifically to target molecules. Using a technique called systematic evolution of ligands by exponential enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, which could bind their targets with high specificity and affinity, were eventually called aptamers, from the Latin aptus, meaning “to fit”. SELEX could be used to classify DNA aptamers as well, and over the course of the next two decades, these nucleotide-based ligand binders would prove to be highly adaptable tools.

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Topics: Plasmids 101, Fluorescent Proteins

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 and the Allen Institute website at

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: Plasmid How To, CRISPR, Techniques

Screening for Successful Genome Editing with Digital PCR

Posted by Guest Blogger on Mar 30, 2017 10:30:00 AM

This post was contributed by Scott Findlay, a Postdoctoral Fellow at the University of Alberta.

If you’re like many researchers these days, you are ready to take (if you haven’t already) the plunge into the world of precision genome editing. When it comes time to (hopefully) validate successful mutation of your favourite gene, there are several different methods available. Thankfully, there are many great resources available to help guide you through the rough waters of mutation validation, such as this "CRISPR 101" post. Next-generation sequencing technologies are the gold standard but they remain cost-prohibitive for many labs, and are often impractical for small projects. Most researchers instead turn to so-called “mismatch nuclease” assays (e.g. Surveyor® or T7E1) for mutation detection. While these methods paved the way for mutation validation, we found these assays frustrating to work with, time consuming, and minimally informative. In this blog post, we’ll introduce digital PCR as an emerging validation technology. Digital PCR has several advantages over mismatch nuclease assays that will be elaborated below

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