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.
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.
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.
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.
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!