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Plasmids 101: Positive and Negative Selection for Plasmid Cloning

Posted by Jennifer Tsang on Aug 22, 2019 8:43:39 AM

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.

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Topics: Plasmids 101, Plasmid Cloning, Plasmids

Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

Posted by Alyssa Cecchetelli on Jun 25, 2019 8:54:52 AM

Horizontal gene transfer (HGT) is the movement of genetic material between organisms. It plays a key role in bacterial evolution and is the primary mechanism by which bacteria have gained antibiotic resistance and virulence. Scientists have studied how HGT occurs in nature and have learned how to introduce genetic materials into cells in the lab.

The introduction of foreign DNA or RNA into bacteria or eukaryotic cells is a common technique in molecular biology and scientific research. There are multiple ways foreign DNA can be introduced into cells including transformation, transduction, conjugation, and transfection. Transformation, transduction, and conjugation occur in nature as forms of HGT, but transfection is unique to the lab. Let’s take a look at these different methods of DNA insertion.

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Topics: Plasmids 101

Plasmids 101: Biotinylation

Posted by Alyssa Cecchetelli on Nov 15, 2018 8:50:12 AM

Biotin and its binding partner avidin are commonly used today in molecular biology for an array of different techniques and protocols. In this post we will discuss the natural role of biotin, biotinylation, the discovery of the biotin-avidin interaction and the uses of biotinylation in molecular biology!

Learn about in vivo biotinylation of bacterial fusion proteins

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Topics: Plasmids 101, Plasmids

Plasmids 101: Codon usage bias

Posted by Tyler Ford on Sep 27, 2018 9:09:41 AM

A similar genetic code is used by most organisms on Earth, but different organisms have different preferences for the codons they use to encode specific amino acids. This is possible because there are 4 bases (A, T, C, and G) and 3 positions in each codon. There are therefore 64 possible codons but only 20 amino acids and 3 stop codons to encode leaving 41 codons unaccounted for. The result is redundancy; multiple codons encode single amino acids. Evolutionary constraints have molded which codons are used preferentially in which organisms - organisms have codon usage bias.

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Topics: Plasmids 101, Plasmids

Plasmids 101: 5 factors to help you choose the right cloning method

Posted by Michael G. Lemieux on Aug 21, 2018 8:31:59 AM

You’ve spent days and weeks thinking of an amazing project. You’ve written your protocols, designed your experiments, and prepared your reagents. You’re going to engineer the best thing since CRISPR; you are ready to clone! But...how?

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Topics: Plasmids 101, Plasmid Cloning, Plasmids

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