Chemical transformation efficiencies

We do a lot of molecular cloning in the lab. Standard practice in the workflow is to make your own home-made chemically competent NEB 10-beta bacteria to be used fresh the day of the transformation. This has worked surprisingly well, and we have made ~ 350 different plasmid constructs in the first ~ 600 days). Each time you do the transformation, it’s important to include a positive control (we use 40 ng of attB-mCherry plasmid) to make sure the transformation step was performing properly (to help interpret / troubleshoot what may have gone wrong if you had few / zero colonies in your actual molecular cloning transformation plates). I’ve done this enough times now to know what is “normal”. Thus, especially for new members in the lab, please reference this plot to see how your own transformation results compare to how it has worked for me, where I typically get slightly more than 10,000 transformants (Note: you may get numbers better than me, which is a good thing!).

Edit 2/24/2022: It recently dawned on me that we haven’t necessarily been using a standard number of bacteria per transformation (ie. we typically prep the same amount of bacteria, and that bacteria normally just gets split into however many transformations are being done at the time. Thus, if there are 10 plasmids being transformed, the number of bacteria per transformation is ~ one fifth of the number of bacteria for when two plasmids are being transformed.

Well, since I had a bunch of data recorded for our positive control transformations (40ng of attB-mCherry plasmid), I decided to see what number of bacteria we had used and if there were any patterns in the transformation efficiencies:

So it looks like, depending on the instance, we used anywhere between 300 million to 10 billion bacteria per transformation. Notably, there was no real pattern in the transformation efficiency, were on average, it worked just as well with the half a billion as it was for 5 billion. Thus, it’s likely that the limiting factor at this point is the DNA and the bacteria are in vast excess.

For those curious, I had previously mentioned that 1mL of our e.coli at an OD of 1 corresponded to 5e8 bacterial cells / colony forming units. Thus, for a 180 mL culture at an OD of 0.2, I would do [5e8 cells/mL at OD1] * [0.2 of OD1] * [180 mL] * [1 billion cells / 1e9 cells] = ~ 18 billion cells. So if we were doing 9 transformations with this culture, it would be about 2 billion cells for each transformation.