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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.

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Topics: Plasmids 101, Plasmid Cloning

FastCloning: A Newer, Simpler Cloning Technique

Posted by Guest Blogger on Nov 1, 2016 10:30:00 AM

This post was contributed by guest blogger Meghan Monroy, a graduate student in Protein Science at the University of Connecticut.

Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without sequence alteration. Molecular cloning techniques are some of the most widely used techniques in the biological sciences and constitute foundational elements of biomedical research. Traditional restriction cloning is one of the oldest of these techniques and is a multi-step process consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely performed by many labs, a variety of other cloning techniques with higher efficiency and simpler procedures have been developed. Some of these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning, and overlap extension PCR. Although each type of cloning has its advantages, most scientists still encounter several struggles with these techniques: unwanted mutations due to excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific sequences for base pair overhangs, insert and vector purification, and, most importantly, excessive time requirements. FastCloning is a simpler yet reliable cloning technique that was developed by Chaokun, et al., in 2011. This method is ligation independent, it does not require purification of insert or vector products, nor does it require the use of specific sequences. Read on to learn how easy this process is and to get tips for applying it in your own lab.

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Topics: Plasmid Cloning

Plasmids 101: TOPO Cloning

Posted by Lianna Swanson on Oct 27, 2016 10:30:00 AM

Toposiomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the basic ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen bonds. This post focuses on "sticky end" TOPO (also called TOPO-TA) cloning; however, the TOPO cloning technique has also be adapted for blunt end cloning.

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

Plasmids 101: Methylation and Restriction Enzymes

Posted by Marcy Patrick on Jun 30, 2016 10:30:00 AM

Have you ever tried digesting with XbaI or ClaI restriction enzymes and gotten unusual or unexpected results? Or considered why DpnI will degrade your template DNA from a PCR reaction but not the newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction digests.

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Topics: Lab Tips, Plasmids 101, Plasmid Cloning

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

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