If you’re into cloning, you’re probably aware that there are several methodologies currently available for approaching it. These include the traditional restriction enzyme/ligase-mediated method, the more recently developed Gibson Assembly Cloning and Gateway® cloning technologies, as well as several others. Each method is unique and relies on specific components that are key to the cloning reaction. Understanding the specific components is essential for choosing the correct cloning method for your own experiments, and here we will focus on a unique gene that makes the popular GatewayTM method possible: ccdB. But what is ccdB, what role does it play in modern cloning, and why should you learn more about it? Read on to find out how ccdB can make your cloning experiments a little easier.
Numbers in the large colored circles are rough approximations of the total number of CRISPR plasmids for that particular organism available at Addgene. Percentages represent the fraction of that total with the indicated function.
One huge reason CRISPR has become such a popular genome editing tool is its developers’ willingness to make their CRISPR technologies available to the academic research community. At Addgene, we’ve helped distribute many of these technologies in plasmid form and are proud to have facilitated their fast adoption. However, in many cases the plasmids themselves are only the starting point for the production of viruses used to deliver CRISPR components to cells or organisms under study. In the past we’ve left the arduous task of virus production to individual labs, but now we’re very excited to provide ready-to-use CRISPR lentiviral preps to researchers across the globe.
Here at Addgene we’re getting into the holiday spirit by kicking off our annual #DeckTheLab contest. The bar is set very high from last year’s impressive entries, but we have faith our community of creative scientists will deliver some fabulous photos again this year!
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, Dalila Cunha de Oliveira.
Bricking Science is an idea built, literally, 'brick-by-brick' to introduce people all around the world to the lives of researchers and PhD students.
Everybody in science knows that there are many ways your experiments can go wrong. Whether it be a bad fridge freezing your samples, or a dysregulated water bath boiling your experiments, just about anything can disrupt your bench work and sometimes no culprit can be found…. In our lab we call this mysterious source of failure the lab gnome.