Recently, when I was doing plasmid curing by incubating E. coli harboring the plasmid in liquid LB at 42 degree. After overnight shaking incubation, there was precipiation formed. When prolonged the incubation, more precipitation was observed. Does anyone know what the precipitation is? Is this fromed by nutrition from the medium under higher temperature, or idead E. coli cells? Thanks.
Dead E. coli!
Is it necessary to grow cells at 42ºC? Most strains do not do too well at 42ºC for a prolonged periods of time.
Thank you guys. I learned from some literature E.coli may be able to lose its plasmid when cultured at 42 degree, that's the reason why I grew them at 42 degree. However, after two times overnight incubation, the plasmid wasn't be cured from E.coli, but precipitation was formed.
If you simply want to cure the plasmid (with antibiotic resistance), let it sit in medium without antibiotics at room temperature for a few days. It will grow slowly but after 2-4 cycles of growth, chances are good that you will lose the plasmid.
If need be, you can replicate colonies on two plates - with and without the antibiotics and identify the colonies without the antibiotics resistance.
Hi again,
Basically Tausif Alam is right but if the goal of the experiment is to chase away a ts plasmid encoding the wt allele of a gene to evaluate the genomic mutant allele of the same gene then it may be complicated if you don't put pressure (especially if the mutant allele has a severe growth defect). If it is what you want to do then put some isolated clones on plates overnight at 42°C rather than culturing cells in liquid then restreak the growing clones at 42°C for e second round and finally test some growing ones for the loss of plasmid (via the loss of the specific marker).
My former lab routinely cured ts plasmids at 42C so E. coli is quite OK at this temperature (on plates for at least overnight incubations). I'm not sure why everyone seems to think 42C is fatal to E. coli.
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