Cosmid miniprep - (Sep/04/2006 )

Hallo,
i read that for the isolation of the cosmid from E.coli, the overnight culture must not grow for more than 12-14 hours...has someone explanation for that??

thanks in advance

Nope, I have no idea. With RecA deficient cells and DH10 cells, there shouldn’t be that much of a problem. Perhaps the cells in your use are RecA+. I hear those guys grow faster (thus used by some labs to do molecular cloning) and express proteins better. Or perhaps the cosmid in question contains some unstable sequence that E.coli don’t like, like large runs of single nucleotides or dinucleotides.

The usual explanation against keeping bacteria cell cultures from growing too long is exhaustion of antibiotics in the culture. Which leads lost of selection and thus the plasmid. The time for antibiotic exhaustion is dependent largely on the number of bacteria in the culture, which in turn depends on temperature and inoculation size. For E.coli growing at 37 Celsius 14hr tops, is a good rule of the thumb. However if the culture were grown at 30 Celsius, 24hr would be okay.

Another reason is low level recombination…even in RecA minus mutants. However I have only seen this in BACs contain tandem repeat arrays and only after the culture had been sitting around for a week.

for large plasmids/cosmids, the bacteria is not very happy with them. Hence, the bacteria need to be in very good condition for maintaining such DNA. If your culture is slight unfriendly, which causes no problem for plasmid preparations, the large DNA start to be degraded and the preparation quality decreases.

For such preparations, a preculture/culture is recommended.
Also i've done 13h culture for 135kpb plasmid extraction with good quality.