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

Tips for CRISPR Gene Editing in Mice

Posted by Guest Blogger on Jun 28, 2016 6:59:27 AM

This post was contributed by guest blogger Samantha Young.

The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers are now using this system in ever more creative ways, (Wang et al., 2013, Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase. But what is the easiest and fastest way to use the system in mice? We'll have a post that goes into the mouse genome editing process in a bit more detail in the coming weeks, but, in this post, we will outline a simple method for selecting the guide RNA, validating its efficacy in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post will help get your in vivo research up and running as soon as possible!

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Topics: Plasmid Technology, Genome Engineering, Lab Tips, CRISPR

Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

Posted by Mary Gearing on Jun 9, 2016 10:30:00 AM

Biologists are going gaga over the newest gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo for short. Addgene has already distributed this plasmid all over the world, and the question on everyone’s minds is: could NgAgo replace CRISPR? Such a drastic shift won’t happen overnight, but there are a few reasons why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more!

Find New Tips in the Han Lab's NgAgo Protocol Here!

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Topics: Plasmid Technology, Genome Engineering, CRISPR

PiggyBac-ing Through the Genome Editing Field

Posted by Guest Blogger on May 31, 2016 11:30:00 AM

Addgene is proud to announce that we recently acquired the ability to distribute plasmids with the piggyBac™ transposon. These plasmids, when combined with a source of piggyBac™ transposase (available from Transposagen or a licensed distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences distributed throughout the genome. We encourage you to deposit your piggyBac™ transposon vectors with us to help us expand this useful resource. While Addgene cannot distribute plasmids with the piggyBac™ transposase itself, please read on to learn more about this exciting technology from the folks at Transposagen.

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Topics: Plasmid Technology, Genome Engineering

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

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