Cloning of plasmid - (Sep/27/2008 )

Hi this is probably a silly question but just want to know for overnight cultures of DH5 alpha E.coli cloned with the plasmid of interest, does it matter whether the time for the culture incubation exceeds the norm overnight/16 hours? If it was to grow longer would it affect the yield of plasmid? as in if it passes the exponential growth phase would it matter?

Thanks in advance!

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If the cultures exceed the 16hrs, the DNA yields can go down significantly.

We normally grow for 12-15 hrs.

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Many people seem to think that growing cells for longer would lead to an increased yield of plasmid DNA, but this is not the case, and in fact, overgrowing your cultures normally leads to a decreased yield.

Once a cell culture exits the exponential phase, cells start lysing and releasing their contents (including plasmid DNA) into the medium. However, you can only isolate plasmid DNA from intact cells. Remember that the first step of plasmid purification is to pellet the cells. If cells have already lysed, their plasmid DNA will remain in the supernatant during this step, and you won't be able to recover it. You only get plasmids from the intact cells that are present in the pellet. The longer you overgrow your culture, the more DNA you'll have in the supernatant and the fewer cells you'll have in your pellet.

Like scolix, I generally use 12-15 hours, and this works fine. Normally the cultures are only somewhat turbid, but the yields are purity are both very good.

Ginger

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OK great thanks guys!

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