Latest Posts

All Posts

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.

Read More >

Topics: Plasmid How To, Plasmids 101, Plasmid Cloning

Pushing the Limits of DNA Assembly

Posted by Guest Blogger on Sep 6, 2017 9:01:33 AM

This post was contributed by guest blogger Lydia Morrison from New England Biolabs.

What is DNA assembly? In the context of cloning, DNA assembly refers to a method of physically joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA assembly available, including: Gibson Assembly®, BioBrick® Assembly, Golden Gate Assembly, and NEBuilder® HiFi DNA Assembly. Gibson Assembly allows the production of scarless DNA constructs using homologous regions to guide the joining reaction. BioBrick Assembly will leave scar regions at the site of fragment joining, but this is fine for its goal of creating a choice of standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly also allows the creation of standardized DNA constructs, but its use of Type IIS restriction enzymes results in scarless assembly. Finally, the NEBuilder® HiFi DNA Assembly method from New England Biolabs® has minimal upfront requirements and allows you to expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments, and generate constructs for producing single-guide RNAs – but it also allows you to skip purification steps and end-repair steps with well-designed fragment overlap sections, while still creating scarless plasmid inserts. 

Read More >

Topics: Techniques, Plasmid Cloning

MXS Chaining

Posted by Leila Haery on Feb 7, 2017 10:30:00 AM

High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences into plasmid DNA. In high throughput cloning techniques, although the specific sequences of the genetic elements may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate each element into the final construct. This strategy can be used to build vectors with diverse functions, and thus, is used in many biological fields. In synthetic biology for example, high-throughput cloning can be used to combine the functions of different genetic elements to generate non-natural tools such as novel biological circuits or sensors. Given the expanding palette of fluorescent proteins and the availability of powerful imaging technologies, the combination of multiple fluorescent protein sequences to develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent reporters can be used to detect structure and protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and Neveu, 2015).

Read More >

Topics: Fluorescent Proteins, Plasmid Cloning

Plasmids 101: Gateway Cloning

Posted by Maria Soriano on Jan 12, 2017 10:30:00 AM

When facing a cloning project, scientists are no longer limited to traditional restriction enzyme cloning. Instead, you can choose a molecular cloning technique that will work well with a given set of resources, time, and experimental needs. Since its invention in the late 1990s, Gateway cloning technology has become very popular as a rapid and highly efficient way to move DNA sequences into multiple vector systems. With the appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety of expression systems. Keep reading to learn more about the Gateway cloning method and its advantages.

Read More >

Topics: Plasmids 101, Plasmid Cloning

Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

Posted by Michael G. Lemieux on Dec 8, 2016 10:30:00 AM

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.

Read More >

Topics: Plasmids 101, Plasmid Cloning

Blog Logo Vertical-01.png

Subscribe to Our Blog