Which is the best method for long term preservation of transformed Rosetta-gami (DE3)pLysS? Will it expel the pET vector with insert, if I keep them at 4°C for a week?
Your cells will not lose their plasmid as long as they are kept in a selection medium with the proper antibiotics (chloramphenicol por pLysS and the selection antibiotic for your plasmid). However, cell do not survive for long in liquid medium, so you should consider keeping a streak of your cells in solid selective medium (i.e. in a Petri dish), for up to 4 weeks.
For long term-preservation you can use two methods: (1) store as a glycerol stock (10 years duration) or (2) store in stab agar (longer than 10 years).
Glycerol stocks are easy to prepare. Just add 1 volume of a late exponential phase liquid culture of your cells in rich selective medium (i.e. with the proper antibiotics added) to 1 volume of sterile 40% glycerol in a cryovial (e.g. Nunc or Nalgene), mix well by vortexing and store immediately at -20ºC to -80ºC.
In these cases I recommend making small overnight stocks at varying strengths of antibiotic selection for each plasmid (normal selective pressure, 1.5x antibiotic, 2x antibiotic, 2.5x antibiotic) and then will make a glycerol stock of the culture that grew at the highest antibiotic concentration and freeze at -80˚C. This worked for me with pRARE2 plasmid and pET28a plasmid for my thesis work. Pelleting these in a microcentrifuge (in aliquats if you like) at max speed for 1 minute, pouring off the media, and freezing at -20˚C can work well too and be thawed and resuspended for innoculations. If you must store it in the fridge, you could try just putting the culture in the fridge (but I've never actually tried this)
I ended up actually purifying out the pRARE2 plasmid from Rosetta-Gami2 cells and have done fresh co-transformations into a BL21(DE3) strain that is also RecA- and EndA- and made glycerol stocks and used this as my expression stocks I still got the extra tRNAs and had improved expression of my eukaryotic gene of interest. You'll lose the "origami" function (improved disulfide bond formation and folding) if you do this though as that's an actual mutation in the host E. coli genome supplied.
A note on the pLysS, it works great for strict regulation of the pET vectors, but you can also try adding glucose to the medium (catabolic repression of the Lac operon) prior to using the specialized plasmids. Sometimes it's sufficient, other times it's not.
Hi Michael, in which cases we might lose the ''Origami'' function? - in term of preservation.
hi
i am trasnforming my gene in Rosetta strain. my genes are in pQE-TriSystem His·Strep 1 vector. i am not getting transformants. can anybody tell me is there any incompatibility between my plasmid and pRARE plasmid??
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