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You are here: Home / Archives for Plasmid preps

CRISPR/Cas9 vector delivery maximized with DNA-In CRISPR

05/11/2015 By Philippe Fixe, PhD Leave a Comment

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.

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Filed Under: Gene Expression - Molecular Biology, Stem Cells Tagged With: CRISPR, CRISPR-Cas9, Genome editing, Genome engineering, Plasmid preps

Which endotoxin testing assay in bioproduction?

29/09/2015 By Isabelle Topin, PhD Leave a Comment

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.

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Filed Under: Gene Expression - Molecular Biology Tagged With: endotoxin, Lab services, LAL, LPS, Outsourcing, Plasmid preps, Rec. protein

5 tips for working with unstable ORF expression clones

26/05/2015 By Mark Livingstone Leave a Comment

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.

Source: http://en.wikipedia.org/wiki/Agar_plate

Source: http://en.wikipedia.org/wiki/Agar_plate

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?

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Filed Under: Gene Expression - Molecular Biology Tagged With: ORF, Plasmid instability, Plasmid preps, Tips & tricks

Why not outsource your plasmid preps ?

29/01/2015 By Frédéric Dubor, PhD Leave a Comment

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.Screen Shot 2014-12-31 at 4.55.17 PM

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.

But it takes time.

[Read more…]

Filed Under: Gene Expression - Molecular Biology, Supplying Discovery Tools Tagged With: Lab services, Molecular biology, Plasmid preps, Routine, Time consuming

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