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plasmid extraction - why dilute (May/05/2010 )

Dear all,

I have the following question.

When doing a plasmid extraction I need to inoculate 5ml broth and grow this the night and then I need to take 1ml of this broth and dilute in it 100ml and grow this overnight to start the extraction.

I wonder why I need to that extra step of starting with 5ml and then diluting it. Why cant I just inoculate the 100ml with my cells from my plates?

-lucilius-

I'm no bacteriologist so I will probably only get this less than half right but:

If you were to inoculate a stab of a plate colony into 100ml, there would be too few bacteria for that size culture and wouldn't reach sufficient density as required for plasmid extraction or whatever in a reasonable time (or at all?).
Also, by this stage many more of your bacteria will be dead as the early ones will have gone through their exponential growth and died off..... (or something.....)

Could it also be to do with bacteria growing better in the company of others? ie like cells in tissue culture grow better when closer to other cells for growth factors and the like? Can anyone help on that??

So in summary- I clearly don't have a very precise idea of why- but it just is......hopefully someone else can clear it up for the both of us :(

-leelee-

I have never done this with a plasmid extraction and neither has anyone I know! I just inoculate 5 ml of broth from an isolated colony on a plate, grow it for 16 hrs, and then spin down the whole 5 ml and extract from the pellet.

lucilius on May 5 2010, 11:59 PM said:

Dear all,

I have the following question.

When doing a plasmid extraction I need to inoculate 5ml broth and grow this the night and then I need to take 1ml of this broth and dilute in it 100ml and grow this overnight to start the extraction.

I wonder why I need to that extra step of starting with 5ml and then diluting it. Why cant I just inoculate the 100ml with my cells from my plates?

-microgirl-

in general culture volumes >10 ml should be inoculated with a pre-culture to get maximal yields, therefore grow cells o/n and dilute the pre-culture 1:1000 in your e.g. 100 ml.

This prevents you from under- or overgrowth of your culture since the best yields are obtained with cells in the late log-phase.

Regards,
p

-pDNA-