Long time ago, I read that the culture for plasmid isolation should be fresh for best results. I took from that that it should be actively growing and was trying to get such cultures since then. But now, I read that the culture should actually reach stationary phase because then it's not actively dividing and thus there is more supercoiled plasmid rather then relaxed one.
I don't often check plasmid on gel, but it's true that when I did recently, I did have about half of plasmid as supercoiled and half as relaxed. So was I doing it wrong for so long?
Hello,
In our lab, we generally isolate plasmid from an overnight bacterial culture using commercial Miniprep kit. The result is good and the DNA yield is higher. I also tried to isolate plasmid from a 6-8 hour old culture. The result is quite fine but the conc. is obviously lower than the overnight culture. I generally check the plasmid by agarose electrophoresis with undigested plasmid and digested plasmid sample with a restriction endonuclease having two restriction sites on the plasmid. I mostly see a higher fraction of supercoiled band on the gel (which migrates faster) than the linear and the nicked bands. Sometimes there is genomic DNA contamination which appears on the band (>10 kb). You can run the plasmid on the gel in two conditions: undigested and digested (with an endonuclease of unique restriction site) for a comparative analysis.
It depend really of what you want to do with DNA (it is said that cells are better transfected with supercoiled DNA) and with "modern" plasmids (with pbs or puc origins) and E. Coli strains (DH5 type) the plasmid yeald is good. For minipreps 3ml overnight culture from a colony picked on a plate is OK, for larger prep= 500ul of this overnight cutlure (kept in the fridge for the time of analysis and even for several days) into 50-100ml of fresh LB and overnight culture then exctraction with a commercial kit. we almost never check the undigested plasmids...
Freshly inoculate bacterial culture into the broth and incubate overnight, as it to be in logarithamic phase for better results
Plasmid extraction involves first selecting a fresh bacterial colony from E. coli strains (DH5 type) and shaking it overnight. The next day, run the PCR and check the bands, if the band is correct, shake the colony into the broth for the same 12 hours overnight. The next day, you can extract the plasmid from it. However, an important tip is to check the broth's color and smell before plasmid extraction.
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