I'm trying to extract plasmid DNA of e.coli and I'm growing a pre-culture in LB agitated medium at 37ºC. I should have an OD(600 nm) 2,6 to start another culture but it only reaches OD 1,16 and then starts to decrease. What should I do?
An OD of 2.6 would be quite high for E. coli unless you are using special media and aeration techniques, or baffled flasks etc. In a normal flask or culture tube I would expect an OD around 1.5-1.8. Also note that you can't actually accurately measure above 1.0 (assuming normal 1cm cuvette) unless you dilute before you measure.
However if your OD is actually decreasing significantly, this might be a sign of some type of lysis, but it probably depends on whether you are seeing slight decline or a significant decline. I would not worry about a slight decline but if significant then I would worry.
Hi Carolina,
In addition to Benedik's answer, after the stationary phase the bacterial growth enters the death phase. So the OD600 reaches until 1,5-1,8 and then declines. Please measure OD every 30 min to get a growth curve and then indicate the culture time for highest OD. Actually, you don't need to arrive OD 2,6 to start another culture. An overnight culture (about 16 hours) is enough to reculture for extracting plasmid DNA. An important point is that what is the inoculation amount to be added into the LB medium. For example, I add 10 microliter thawed E. coli culture to the 100ml LB medium and incubate it for overnight.
Have a good study.
This is a situation where more is not always better. As mentioned above, once the cells reach stationary phase they die. Those dead cells are no good for plasmid extraction. You really want nice fresh healthy growing cells, 12-16 hours is plenty of time. When you are ready for the plasmid prep, use no more than the maximum cell volume suggested in the protocol. More cells can overwhelm the capacity of lysis and cleanup steps.
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