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.
Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation, elongation, and termination. In a previous post we discussed the promoter's role in the initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination. Read on to learn more!
One of the best things about sharing plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through our quality control processes. Every plasmid we receive is rigorously verified before becoming available to the community.
This is no small task, however, at a repository that consistently receives around 200 new DNA samples every week. Here we will provide an inside look at the steps we take to verify the identity and quality of the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids.
In a previous Plasmids 101 blog, we reviewed the salient features of several popular strains of E. coli for DNA propagation. While great for cloning purposes, these E. coli strains are not usually well suited for recombinant protein expression. Many challenges can arise when over-expressing a foreign protein in E. coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular commercial strains designed to avoid them.
Homologous recombination is the process by which nearly all domains of life repair genomic damage, specifically double strand breaks. Researchers have long taken advantage of this natural process to integrate protein tags into the genomes of S. cerevisiae and S. pombe. The protocol is surprisingly simple, requiring only a PCR product containing the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal site of insertion. The linear PCR product is introduced into the cell by direct transformation. A given insert will typically contain both a protein modification sequence and a selectable gene product for isolation of successful transformants.
Addgene distributes several ready-to-use, modular plasmids, combining fluorescent tags, epitope tags, protease sites, and selection markers. These are especially useful in protein complex studies where tagging of multiple protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated. Simply design your amplification primers with the desired targeting homology—in frame, of course—and start tagging!