So I have noticed for a while that for the same plasmid, sometimes it can be seen in a gel in only one conformation, often two and sometimes three or more.
Sometimes samples of the same plasmid which have been purified at the same time and stored in similar conditions have different conformations.
Today I noticed that a high concentration purification (200+ ng/ul) was predominantly in the conformation with the highest electrophoretic movility (out of 2) while the same plasmid at a lower concentration (60 ng/ul) was predominantly at the conformation with the lowest electrophoretic movility.
(I assume those conformational classes might be circular and supercoiled respectively)
Does the concentration of plasmid affect conformational balance? what other environmental factors may affect this in a in vitro (lab) setting?
I mean, temperature is a given; but I think I have noticed that not just current temperature may affect conformation; but also the order of temperature changes (if it was cooled down a few hours or heated before reaching current room temperature or fridge temperature).
Thank you in advance
One factor may be that most purification methods involve a denaturation step useful in separating genomic dna from plasmid dna. This will not be the most stable supercoiled configuration and may lead to nicking and some degradation
As Paul Rutland mentioned, often the purification step is the biggest variable in whether your plasmid is supercoiled or nicked. But even with a predominantly supercoiled plasmid prep, over time you will see a slow conversion to nicked. This can be caused by light damage, freeze thaw cycles, or random events accumulating over time (random events meaning we don't really know the cause)
Maintaining room temperature for plasmid conformation is one of the crucial environmental factors. The temperature should be 20-25 degree celcius, which is suitable for plasmid conformation.
The postharvest softening and corresponding quality deterioration of blueberry fruits are crucial factors that hinder long-distance sales and long-term storage.
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