Hi, I am wondering if different plasmid extraction kits yields different quality of extracted plasmids, which subsequently affected some downstream analysis such as nucleofection in human embryonic stem cells. I am asking specifically for the used in human embryonic stem cells context.
Could you also provide your preference and the reasons?
Thank you.
Human embryonic stem cells are not the focus of my current research. However, regarding plasmid extraction, different kits may yield varying quantities and qualities, but generally, they are capable of satisfying downstream transfection experiments. The ability to remove endotoxins is an important factor to consider when choosing a kit.
As mentioned Wizard® Plus SV Minipreps DNA Purification kit from Promega is one of the best kits for good quality AND quantity plasmid isolation with a close competition from the NEB's Monarch kits and favorogens plasmid miniprep kits. And also as mentioned above use an endotoxin removal based kit for your work.
Kits vary with their plasmid recovery based on mainly the silica bead packing, the thicker bed columns do have the potential to bind a lot of DNA but they require a lot more elution volume as well and the plasmid gets highly diluted. Thus, columns with thinner beds in my experience seem to give best results.
One strange thing I have also observed is that when I left columns at 4 degrees for some time and then used them they gave me a better yield, I am unaware of the reason or whether this phenomenon is actually true or not but since it seemed to work for me I can suggest this as well.
One final thing incubating the bacterial cultures on ice for 1-2 hrs before plasmid isolation seems to improve the Superhelical plasmid yield as well. Maybe because the DNA repair/ packing mechanisms getting activated during the dormant growth stage in bacteria.
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