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!
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?
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.
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 make specific DNA sequence changes to the genome and to perform genome-wide functional screens to identify genes involved in a phenotype of interest. This blog post will discuss the differences between these approaches, as well as provide updates on how best to design gRNAs for your experiments. You can also find validated gRNAs for your next experiment in our Validated gRNA Sequence Datatable.
One of the best things about sharing plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through our quality control processes. Every plasmid we receive is rigorously verified before becoming available to the community.
This is no small task, however, at a repository that consistently receives around 200 new DNA samples every week. Here we will provide an inside look at the steps we take to verify the identity and quality of the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids.