What is the E coli transformation efficiency for 2 plasmids? Are any studies that have looked at the correlation between number of plasmids transformed and transformation efficiency? Is there a commercial E coli strain that is better at co-transformation?

For example, electroporation with single plasmid yields around 1 × 10^10 cfu/μg. I would like to know what is the limit for co-transformation and whether it can be or has been improved.

  • $\begingroup$ Nothing to back this up with so just a comment: my understanding is that the cells which are competent take up a lot of DNA, so cotransformation is easy to achieve as long as you can select for both plasmids (if they have replication/segregation features from the same incompatibility group they will segregate rapidly). I once did a Tn5 mutagenesis experiment which required propagating the plasmid in a Tn5 strain, preparing plasmid, transforming a recipient at high DNA input and selecting for kanamycin resistance. Many transformants contained more than one plasmid but they resolved easily. $\endgroup$
    – Alan Boyd
    Mar 29, 2014 at 13:01
  • $\begingroup$ The efficiency is pretty low. You have to be able to select for both plasmids individually (for example plasmid 1 with ampicillin and plasmid 2 with choramphenicol or other possible antibiotics) otherwise the cells will rapidly loose one of the plasmids. Usually the shorter and less "energy expensive" stays, if it maintains the selection marker. $\endgroup$
    – Chris
    Mar 30, 2014 at 18:13
  • $\begingroup$ I've always understood for this process to be incredibly low. When you consider how much DNA is added and how many competent cells there are, if you have even 1% transformation efficiency, you'll always get lawns. Since we don't, that gives a rough estimate of the process. $\endgroup$
    – bobthejoe
    Apr 3, 2014 at 2:38
  • $\begingroup$ I also make the previous comments into a nice answer. $\endgroup$
    – Chris
    Jul 29, 2014 at 16:21

1 Answer 1


There is no problem to transform bacteria with more than one plasmid. The usual methods (either chemical competent cells or electroporation) are working fine, although electroporation might give you better results, as the cells take up more DNA. On the downside you have to make sure that your cells are prepared nicely, otherwise the electroporation cell will produce a short circuit (of course chemically competent cells can be problematic, too).

What you will definitely need are two (or more) different selection markers. The easiest will probably be different antibiotics (something I used in the lab and which works nicely) or other like genes which provide essential substances for the cells. As long as the selection pressure on the cells is held up, both plasmids will stay in the cells, when you take away one of them, the corresponding plasmid will get lost within a few days.

Regarding the efficiency of co-transformations I haven't found any papers. There is however, some data available on factors which influence the effiency of single transformations and I don't see, why these shouldn't be applied for co-transformations as well.

First, there is the size of the plasmid. The bigger the plasmid is, the lower gets the transformation efficiency. The figure from Inoue, 1990 is pretty clear in this point:

enter image description here

So for this reason I would use plasmids as small as possible.

Second, the density at which the growing bacterial culture which is used to prepare the competent cells is harvested has a big influence on the transformation efficiency. Tang and colleagues (1994) found out that growing the cells to a higher density than the classical OD$_{600}$ of 0.6 (which for example Inoue recommends in it paper cited above) is not a very good choice in terms of transformation efficiency. They found an OD$_{600}$ of about 0.94 working much better, see the corresponding figure:

enter image description here

And finally the amount of competing DNA is a big factor. Hanahan, 1983 found out that the transformation efficiency gets lower, the more competing DNA is present:

enter image description here

So I would try to use as little of each DNA to work against this effect.

  • $\begingroup$ Thanks, Chris, but I was more wondering how high with efficiency (number of clones per ug DNA) you can get with co-transformation. For example for single electroporation the efficiency is around 1 × 10^10 cfu/μg so I would like to know what is the limit and whether it can be improved for co-transformation. $\endgroup$ Jul 29, 2014 at 18:51
  • $\begingroup$ I haven't found any papers looking on this for co-transfections, but some data for single transfections. We can do some interpolation from them, I will expand my answer. $\endgroup$
    – Chris
    Jul 29, 2014 at 20:02

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