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

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: Plasmid How To, CRISPR, Techniques

Plasmids 101: Knockout/Knock-In Plasmids

Posted by Benoit Giquel on Dec 1, 2016 10:30:00 AM

One of the most powerful strategies to investigate a gene's function is to inactivate, or "knockout", the gene by replacing it or disrupting it with an piece of DNA designed in the lab. Specially constructed plasmids can be used to replace genes in yeast, mice, or Drosophila through homologous recombination. The concept is simple: deliver a template with a modified version of the targeted sequence to the cell which will recombine the template with the endogenous gene. Here, we'll describe the techniques and the plasmids used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based knockout/knock-in systems, these systems remain valuable, especially in cases where CRISPR cannot be used (e.g. there are no suitable PAM sequences nearby or your gene of interest is difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a knockout strategy!

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Topics: Plasmid How To, Genome Engineering, Plasmids 101

Using Phosphoserine to Study Protein Phosphorylation

Posted by Guest Blogger on Jun 23, 2016 10:30:00 AM

This post was contributed by guest blogger Natalie Niemi, a postdoctoral fellow at the Morgridge Institute for Research in Madison, Wisconsin.

It is commonly cited that approximately one-third of cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array of model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent than previously appreciated. PhosphoSitePlus, one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of any of these phosphorylation events on the activity of a given protein?

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Topics: Plasmid How To, Synthetic Biology, Lab Tips, Techniques

Plasmids 101: Colony PCR

Posted by Guest Blogger on May 12, 2016 10:30:00 AM

This post was contributed by guest blogger Beth Kenkel, a Research Assistant in the Department of Pediatrics at the University of Iowa. If you're interested in guest blogging, let us know!

Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however colony PCR can accomplish the same thing in less time and for less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze product size. This blog post discusses some of the key things to consider when performing colony PCR.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

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