I have just purchased a 12 rxn C43 competent cells kit.
I want to make a stock from it and store in -80 .
After i regenerate the cells by plating them out and culturing in liquid medium, to restore competency can i follow any protocol of E.coli competency and has anybody come across a study comparing protocols, because while similar they differ in small details , or from your own experience and concluded on a mot efficient protocol?
Much apreciated,
Cheers
I have had brilliant results with a two step rubidium chloride method with a number of strains.
The Chan publication I've linked is the only one I know of right off hand that compares different methods of chemical transformation as well as different strains.
The second publication(Inoue) is the method I've used the most. I like it because it simplifies the transformation buffer quite a bit from some of the other protocols I've seen and seems to work well for every strain I've tried it on. Then I've also linked a more easy to follow protocol version of that method.
However, I usually do make one modification: Instead of bothering with the complicated sub-culturing I typically just inoculate straight from a freezer stock into 25 mL SOC in a 50 mL conical tube, then incubate it with shaking overnight at room temperature until it looks like it's around an OD of 0.5ish. Then I scale all the other reagents down accordingly - The protocol given makes a lot of competent cells and I don't typically need to make 400 reactions worth.
https://www.princeton.edu/genomics/mcclean/protocols/Inoue-Method-for-Preparation-of-Competent-E-Coli.pdf
Article Inoue, H. , Nojima, H. & Okayama, H. High efficiency transfo...
Article A comparison and optimization of methods and factors affecti...
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