Transformation Efficiency

Transformation efficiency is a measure of the competency of a cell and its ability to uptake exogenous DNA. Cells can be made competent by growing cells to log phase, holding at 4oC, and washing away media with 50 mM CaCl2 (divalent cation). The standard method of reporting transformation efficiency is:

Transformation Efficiency = # transformants /g plasmid DNA

Therefore, to calculate this you simply need the number of transformants and the amount of DNA that gave rise to those transformants. The number of transformants can be easily determined simply by counting the number of colonies growing on selective media (plus amp plates). The amount of DNA giving rise to those colonies can be determined by asking how much DNA was ‘plated’ on the plate you just counted. This can be determined by knowing the starting concentration of your transforming DNA, the amount you pipetted into your competent cells, the dilutions you accomplished along the way toward plating that plate, and the volume of culture you plated onto the plate. Let’s follow the DNA concentration through the process of a typical transformation.

Pipet 10 L of a 1 ng/L DNA solution into 100 L of compent cells.

How much DNA did you apply to your competent cells?

10 L * 1 ng/L = 10 ng DNA

Incubate on ice one hour, heat shock, add media to a final volume of 1 mL, incubate at 30oC, 30 minutes to allow for expression of the amp resistance gene.

What is the concentration of our DNA at this point?

10 ng of DNA is now in a volume of 1 mL. Therefore:

10 ng DNA/mL is our current DNA concentration.

We now have transformed cells and can plate them out on plus amp plates. Since we don’t know the concentration of these transformed cells, we will accomplish a serial dilution and plate. Let’s perform 3 ten fold dilutions of our transformed cells and plate 0.1 mL of each dilution onto a plate. Now follow the DNA concentrations through this process. We know our DNA concentration in our competent cell mix was 10ng/mL. After one ten fold dilution that concentration would be 1 ng/mL. Another ten fold dilution will make it 0.1 ng/mL and a subsequent ten fold dilution would make it 0.01 ng/mL. We then plate from these tubes 0.1 ml and incubate.

How much DNA was ‘plated’ onto each of these 3 plates?

Plate 1 from our first 10 fold dilution:

The concentration of DNA in that tube was already determined to be 1ng/ml. We plated 0.1 mL onto that plate, therefore:

1ng/mL * 0.1mL = 0.1 ng DNA applied to that plate.

Plate 2: 0.1ng/mL * 0.1mL = 0.01 ng DNA

Plate 3: 0.01ng/mL * 0.1mL = 0.001 ng DNA

Suppose plate 1 yielded 2500 colonies, plate 2 yielded 250 colonies, and plate 3 yielded 25 colonies. Which plate are you going to count? Plate 2, right?

So, what do we know now? We have the number of transformants on the plate and the amount of DNA that gave rise to those transformants.

250 transformants/0.01 ng DNA.

The convention is to report this figure as transformants/g DNA so we must convert.

Transformation Efficiency = 250 transformants/0.01ng DNA * 1000ng/1g

= 2.5 * 107 transformants/g plasmid DNA