During the process of plasmid purification and quantification, which step is the most critical to the success of the process?
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$\begingroup$ What scale are we talking about? Because Giga preps are different from mini preps. $\endgroup$– user137Sep 17, 2014 at 15:36
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$\begingroup$ It's for a mini prep. $\endgroup$– frenchwhorneSep 17, 2014 at 15:38
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$\begingroup$ Dear friend, there are many ways to extract plasmid. Which ones are you talking about? $\endgroup$– M007Sep 17, 2014 at 16:01
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1 Answer
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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.
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$\begingroup$ Just to add some details, don't lyse for more than 5 minutes, have the neutralization buffer ready to go so after you get your last sample lysed and mixed immediately add neutralization buffer to the first samples. When mixing, it's only ok to vortex the cell pellet prior to lysis, after adding lysis buffer, ONLY MIX BY INVERTING THE TUBE. Vortexing after this point can shear DNA. If you overdry the pellets, you can resuspend them by leaving them in the TE buffer for several minutes at 37C. And don't do too many samples to handle them all, with practice you can do 24 samples without trouble. $\endgroup$– user137Sep 17, 2014 at 16:05
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$\begingroup$ Thanks for this additions. More than 24 is usually not possible, since this is the limit for most benchtop centrifuges. Some even take up only 18 tubes. $\endgroup$– Chris ♦Sep 17, 2014 at 16:13
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$\begingroup$ So, in summary, all of the steps are critical. $\endgroup$ Sep 17, 2014 at 18:08
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$\begingroup$ @AlanBoyd You can loose yield in all of them. I wouldn't say one is much more complicated than others. $\endgroup$– Chris ♦Sep 17, 2014 at 18:11
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1$\begingroup$ @Chris I agree, I was trying to stress to the OP that the whole process should be carried out carefully, which is what you said. Remembering to add antibiotic to the cultures is also critical! $\endgroup$ Sep 17, 2014 at 18:17