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Plasmids 101: Optimizing Plasmid Yields

Posted by Julian Taylor-Parker on May 26, 2016 10:30:00 AM

Most of the time, plasmid prepping is a breeze. You get your stab from Addgene, streak for single colonies, sub-culture, and prep with one of the many commercially available DNA prep kits or your lab's favorite in-house protocol. DNA yields for this procedure are typically in excess of 100 ng/ul, more than enough DNA to proceed with most applications, such as PCR, cloning, transfection, or long-term storage. But what about those pesky situations where your plasmid yield is sub-optimal? If you have already purifed your plasmid, you can try to concentrate the DNA using a speed-vac, ethanol precipitation, or other chromatographic methods. But wouldn't it be nice to avoid an extra concentration step? If you are consistently getting sub-optimal plasmid yields from your prep, you may want to consider optimizing your growth conditions. In this blog, we will outline many of the variables that could affect DNA yields and suggest steps to super-charge your plasmid preps.

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Topics: Lab Tips, Plasmids 101

Plasmids 101: Colony PCR

Posted by Guest Blogger on May 12, 2016 10:30:00 AM

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.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

Plasmids 101: FLEx Vectors

Posted by Michelle Cronin on Apr 28, 2016 10:30:00 AM

In a previous post from our Plasmids 101 series, we learned how the Cre-loxP recombination system can be used to induce site-specific recombination events, and that the orientation of the flanking loxP sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of both wild-type and mutant loxP sites has allowed scientists to leverage this system in new, creative ways. Today’s post will focus on one such strategy--the FLEx switch--which utilizes recombination elements to turn off expression of one gene, while simultaneously turning on the expression of another!

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Topics: Plasmid Technology, Plasmids 101, Cre-lox

Plasmids 101: Terminators and PolyA signals

Posted by Julian Taylor-Parker on Mar 31, 2016 10:30:00 AM

Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation, elongation, and termination. In a previous post we discussed the promoter's role in the initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination. Read on to learn more!

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Topics: Plasmid Elements, Plasmids 101

Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

Posted by Brook Pyhtila on Mar 1, 2016 10:30:00 AM

Over the past decade, scientists have developed and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have become more and more common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on. In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to put together DNA fragments.

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Topics: Plasmid Technology, Plasmids 101, Protocols, Plasmid Cloning

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