For maxipreps, cant you just add all the stuff that would be in P1 (RNase A, EDTA) then just add P2? Because if we pellet cells then resuspend theres got to be a reason, right? Is it slats and other stuff in the media that mess with lysis?


The reason this centrifugation/resuspension step is done has a simple reason: It concentrates the bacteria in a much smaller volume which is much easier to handle afterwards.

For a maxiprep you typically have a culture volume between 250 and 500ml, depending on the copy number of the plasmid. After centrifugation the pellet is resuspended in 10ml of Buffer P1, for lysis another 10ml of buffer P2 are added, for neutralization 10ml of P3.

Imagine doing this with 500ml culture volume - adding 500ml of each P2 and P3. This might work for a miniprep, but definitely not for larger volumes. Additionally, the centrifugation steps are carried out at high g numbers - typically something around 15.000xg. Doing this with this really high volumes is complicated and needs big centrifuges, which a lot of people are not trained anymore to operate or have available.

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    $\begingroup$ I want to add that there are some kits that allow direct addition of the "lysis buffer" to the cell suspension in the medium. Of course, as you said, yield would be low. $\endgroup$ – WYSIWYG Jan 8 '19 at 12:54

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