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):
- 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.
- 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.
- 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.
- 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.
- Drying: Overdrying of pellets can cause problems when you try to dissolve them again.