I was reading some old description of the protocols used for the transformation of bacterial cells.

In the description I read that the transformation works best with low amount of DNA, and if we increase the amount of DNA used the amount of transformant cells does not increase proportionally.

I just wonder why, since in my eyes the process of entering the cell is mainly related to the diffusion principles, and therefore concentration dependent.

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    $\begingroup$ If there are many times more plasmids than cells, increasing their concentration when the cells are already saturated would therefore not increase the transformation ratio. $\endgroup$ – March Ho Jun 1 '15 at 8:58
  • $\begingroup$ Thanks, but then it is similar to a enzymatic kinetic. So in theory I will have an increased transformation at higher concentration of plasmid till a saturation point that, I would guess, depends from the strain I use to transform? $\endgroup$ – efrem Jun 1 '15 at 9:01

According to the Hanahan transformation protocol (one of the highest competencies published), a transformation of 200µl will contain $1×10^8 - 1.7×10^8$ cells.

Assuming that the pBR322 plasmid is used, 1ng of plasmid will convert to 0.36fmol of plasmid, or $6×10^8$ plasmid copies.

As we can clearly see, even at very low quantities of DNA used, there are multiple plasmids per cell. Since transformation is thought to be mediated by DNA channels, massively increasing the quantities of DNA would not equally increase transformation efficiency once all of these channels are saturated.

The example from NEB Turbo DH5α cells seems to show this quite well, that once DNA quantities exceed 1ng, the transformation efficiency drops precipitously.

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This being said, if the amount of plasmids available is very large, there is no reason to avoid adding more than 1ng of DNA, as they would not reduce the total number of transformation colonies, but only the efficiency.

  • $\begingroup$ Thanks very much for your nice answer. I am thinking though that in the example you do there will be around 6 copy of plasmid available per each cell. For my understanding there could be hundreds of possibilities (e.g.DNA channels, electroporetic generated holes) for the DNA to go in. SO in theory if we create a saturation curve we can see that the number of transformant is increasing with the amount of plasmid we add, at least at the beginning. When saturation is reached he amount of DNA is not influent. It would not make sense to add more DNA from the start to have higher transformant? $\endgroup$ – efrem Jun 1 '15 at 13:09
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    $\begingroup$ @efrem I am not sure if Michaelis-Menten kinetics (in the sense of the saturation curve) apply in this case due to the mix of many different means of plasmid entry, but the general concept of channel saturation should still apply. $\endgroup$ – March Ho Jun 1 '15 at 13:38
  • $\begingroup$ thanks! So the efficiency drops, but the number of transformed cells increase exponentially. Therefore, if someone is not limited in terms of plasmid amount, and has cells that do not transform so well, it would be better to increase the amount of plasmid to get a higher number of transformant, even though with less efficiency. $\endgroup$ – efrem Jun 1 '15 at 13:45
  • $\begingroup$ @efrem The increase is most notably NOT exponential. It is in fact worse than linear, considering the X axis is log but the Y axis is linear. However, in most cases of bacterial transformation, you are using high concentration plasmids in any case. I can't think of a way to reduce the number of total transformation colonies by adding too much DNA. $\endgroup$ – March Ho Jun 1 '15 at 13:48
  • $\begingroup$ right, sorry. It is indeed linear. Anyway, it seems to me that there is not any major drawback in adding too much DNA (plasmid), in terms of total number of transformed cells. $\endgroup$ – efrem Jun 1 '15 at 13:56

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