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.
Born to a family in Malawi that pushed science education, Kingdom Kwapata grew up to be one of the best of the best students in his home country. He was selected as one of 600 scholars among 100,000 eligible to attend the University of Malawi where he focused on agricultural engineering through biotech. After graduating with a masters of science in Horticulutre Biotechnology, he did his PhD in Plant Breeding, Genetics, and Biotechnology at Michigan State University with the backing of a prestigious Fulbright Fellowship. Dr. Kwapata now works as a lecturer in molecular genetics and biotechnology for Horticulture and Forest Crops at the Lilongwe University of Agriculture and Natural Resources. He recently received an award from nonprofit and Addgene collaborator, Seeding Labs, for lab equipment that will help accelerate his research. Listen to learn more about Dr. Kwapata’s current work and thoughts on the need for more scientific collaboration across the globe.
Lentiviral vectors are one of the most popular and useful viral vectors in the lab. Advantages of lentivirus include a large genetic capacity and the ability to transduce both dividing and non-dividing cells. Lentiviral vectors are the vector of choice for many CRISPR applications, and they’ve also had success in clinical gene therapy applications. Read on to learn more about the current (and future) applications of lentiviral vectors!
Topics: Viral Vectors
This post was written jointly by Addgenies Brook Pyhtila and Nicole Waxmonsky
Resource sharing shortens the time needed to go from planning an experiment to performing one. At Addgene, over 120 labs have deposited CRISPR reagents, including many gRNA-containing plasmids (McDade et al, 2016). Many of the gRNAs contained within these plasmids have been used successfully in peer reviewed articles. If you’re targeting your favorite gene with CRISPR, using one of these validated gRNAs can save you the time that would be spent making and testing entirely new gRNA designs. You can now easily find many validated gRNAs in our newly curated Validated gRNA Target Sequence Table.
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.