Since bacteria naturally contain plasmids, when you do a cloning experiment and purify the plasmids at the end, do you get plasmid naturally present in that bacteria in addition to your vector? The standard plasmid purification kits (miniprep, maxiprep, etc.) aren't sequence specific in their purification (they just seem to isolate all plasmids in the cells). I would assume you want a constant purity of vector. Otherwise, for example, you could have 2 vials of plasmid at 1 ug/ uL plasmid while one has 0.4 ug/ mL vector and another 0.8 ug/ mL vector which wouldn't be good for reproducibility. Or is it that the bacteria used for cloning are devoid of naturally present plasmids which would fix this issue?

  • $\begingroup$ Very interesting question, Chris. What result do you see when you analyze a miniprep from non-transformed bacteria on an agarose gel? $\endgroup$ – imalipusram May 23 '20 at 4:05
  • $\begingroup$ I'm a bit newer to this area of biology techniques so I haven't looked at many plasmid preps yet. The plasmid prep that I have seen on a gel had one band (or 2 bands if cutting the insert out with a restriction digest). If this result is generalizable, it would suggest the vector was present in much greater quantity than any of the endogenous plasmid (if there was any endogenous plasmid at all). I can think of 2 reasons for this (if the result is generalizable): $\endgroup$ – Chris May 25 '20 at 22:47
  • $\begingroup$ Sorry I accidentally posted the previous comment before it was finished. I was going to say the 2 possible reasons that I could think of are (I don't know which reason is the primary reason though): 1) The strains used for cloning have less endogenous plasmid. 2) The bacteria had to express a lot of vector to get sufficient antibiotic resistance such that the vector was present in much greater quantity than you would normally see in endogenous plasmids. $\endgroup$ – Chris May 25 '20 at 23:05

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