Artificial intelligent assistant

What is the most critical step of plasmid purification? During the process of plasmid purification and quantification, which step is the most critical to the success of the process?

There are a few critical steps (which sounds horrible if written together, but the method per se is robust and usually without problems):

1. Resuspension of the pellet: Make sure it is really resuspended and not floating around as a big blob. If it is not resuspended properly your yields will go down dramatically.
2. NaOH/SDS-Lysis: Don't lyse for too long as you may end up with low quality DNA. This denatures the plasmid and makes it often useless for subsequent enzymatic reactions.
3. Mixing: Don't mix too harsh once you lyse the cells, otherwise you may break up genomic DNA of the bacteria which subsequently stays in solution and is not precipitated during neutralization.
4. Neutralization: Make sure you mix good here (by inverting the tubes 4-6 times) but not too harsh. It is critical to neutralize the whole lysate, otherwise proteins may stay in solution.
5. Drying: Overdrying of pellets can cause problems when you try to dissolve them again.

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