I am looking for a very detailed step-by-step protocol for performing stability check of recombinant plasmid DNA in Lactobacillus? Thanks in advance!
Hi Edward,
Well, there might be as much protocols as researchers investigating plasmid satbility !!
What I proprose to you is not the easiest but the more robust approach.
Always proceed from a liquid culture of your transformed bacteria just after transferring them in a non-selective medium (never never from colonies on a solid medium). At regular times, take up an aliquot of this culture and dilute it to get roughly a cell density of about 1000-10000 cells per ml. Spread 100 microliter of this final dilution on a nonselective medium Petri dish (to measure the living cells) and another 100 microlietrs on a selective medium plate (to measure those yet harboring the plasmid). Follow the decay kinetics over hours.
Good luck!
Best wishes
Prof Philippe Urban
Hello Professor Philippe,
Thank You. I know there are a lot of protocols. I've read several papers in LAB (Lactobacillus and Streptococcus spp.), however, the materials and method section are always short, concise and vague. I really appreciate your response.
I will study, try and get back to you with what I come up with soon.
Sincerely,
Edward Alain
Hello!
Did you find a reliable method to measure plasmid stability? I'm interested, too.
Sincerely,
Gábor
Hi,
In cases where your expected stability is very poor (below 1%) I'd suggest to avoid replica plating of say, 100 colonies on non-selective and selection media. Instead spread 100-200 cells on non-selective media, pick each colony individually and patch on selection and non-selection plates again. This way you'll get rare stable isolates (can do for up to a thousand colonies) and it will also give you better counts for assaying the final stability rate.
Good day!
Lakshmi
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