CRISPR-Cas9 is a popular method that brings researchers endless experimental strategies to create their own research-based cellular models. In this post we’ll review a new transfection reagent especially engineered to maximize Cas9 vectors deliveries inside cells with low cellular toxicity.
Pharmaceutical, biotech and medical device industries apply restrictive quality systems in their bio-production processes. Among the numerous parameters controlled daily by scientists, I have selected a particular one for this post, i.e endotoxin detection.
Mammalian expression constructs for certain genes such as human c-KIT are notorious for undergoing frequent recombinations during cloning and transformation steps in molecular biology labs. Experts suggest that certain genes are “toxic” to bacteria thus leading to a situation in which recombined plasmids are favored. While the molecular mechanisms for this toxicity may be unclear, the end result is that efforts to amplify, subclone, mutate, or make derivative vectors often result in a new plasmid with unwanted sequence errors.
Online science discussion forums such as ResearchGate include a variety of strategies proposed by researchers experiencing this kind of plasmid instability. Suggestions include advice such as culturing the bacteria at room temperature rather than 37°C, culturing on plates rather than in flasks, using low copy number EPI400 competent cells, picking the small colonies rather then the large ones from the LB plate, replacing the ampicillin resistance cassette to prevent satellite colonies (so you can see the small colonies), or using a Gateway vector.
But which methods work best?
Plasmid preps are to science what salt is to cooking. It’s so common that nobody notices, but nothing can be done without. There is no mention of this step in any published paper, nobody mentions them in any activity report, no comments are made on time spent on it.
But let’s face it: it takes time, as simple and as usual that it is. Some days spent, here and there. It’s so well planned in your weekly agenda, it’s such a habit, that you don’t even think about it. It’s just routine. When you’ve done a plasmid prep a couple of times, there’s nothing new to learn about it. It’s like riding a bike, once you know you don’t even notice any more.