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This post was contributed by Oskar Laur, head of the custom cloning core at Emory University, and Paolo Colombi, a product development scientist at Addgene.

Cloning can be quite an arduous process. The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies screened might not contain the correct plasmid. There’s a lot that can go wrong! With all the steps in the cloning process, there are also many ways to troubleshoot the cloning experiment. Here are some tips that will help you with your cloning project, and hopefully obtain your coveted plasmid with no substantial delays.

Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here. 

You’ve worked hard to purify your gene of interest, get it into your plasmid backbone, and zap the mixture of DNA into cells. Unfortunately, not every cell successfully takes up plasmid DNA. Among those that do, some now have plasmids that contain your gene of interest, but others will uptake plasmid backbones that re-ligated back on themselves.

Therefore, your cloning strategy needs to identify cells containing the plasmid construct you’re seeking. Fortunately, there are many ways to do this involving positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection in this blog post, but don’t worry, we’ll cover screens in a future post.

This post was contributed by Jacob Lazarus, a postdoctoral researcher at Harvard.

There’s an astounding number of ways to create chromosomal mutations in bacteria, so many that it may be difficult to decide which path to take. A quick and easy way to introduce a mutation in the chromosome is to disrupt expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome, sometimes interfering with expression of surrounding genes. However, there are ways to create scarless mutations, ones that don’t leave any undesired scars in the chromosome.

Microbiome studies have traditionally fallen into studies of who’s there and what are they doing. To address these questions, biologists often use next-generation sequencing. Sequencing the 16S rRNA reveals the identity of the organisms present while sequencing of all transcripts gives clues into what the microbes are doing.

But aside from sequencing, scientists can also study the microbiome by using engineered genetic tools and reporter microbes. Let’s take a look at a few of these methods below.