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Topoisomerase 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 ...
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 ...
In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest. You can use similar processes to add overhangs to your ...
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 ...
When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new ...
If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not only does this system not use site-specific recombination, it also doesn’t require a ligation step! Based on the robust system of homologous recombination found in ...
Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful within both the synthetic biology and genome engineering fields. We’ll walk you through how ...
