One loopful (10 ul) of plasmid DNA with a concentration of 10 ng/ul was used. The cells were incubated in a total of 500 ul of solution, and 100 ul of this solution was used to inoculate the plate. If the transformation efficiency was supposed to be 1048 colonies/ug, explain whether the transformation was successful.
Calculate transformation efficiency from the results of the plate pictured: One loopful (10 ul) of plasmid DNA with a concentration of 10 ng/ul was used. The cells were incubated in a total of 500 ul of solution, and 100 ul of this solution was used to inoculate the plate. If the transformation efficiency was supposed to be 1048 colonies/ug, explain whether the transformation was successful
The Correct Answer and Explanation is:
Here is the calculation of the transformation efficiency and an explanation of the results.
Calculated Answer
Transformation Efficiency: 1.05 x 10⁴ colonies/µg
Conclusion: The transformation was not successful as it did not meet the target efficiency of 10⁸ colonies/µg.
Detailed Explanation
Transformation efficiency is a critical metric in molecular biology that quantifies the effectiveness of introducing foreign plasmid DNA into competent host cells, such as bacteria. It is expressed as the number of transformed colonies generated per microgram (µg) of plasmid DNA used. A high transformation efficiency is crucial for experiments like creating DNA libraries or complex genetic engineering.
The calculation involves three main steps:
1. Calculate the total mass of plasmid DNA used in the transformation.
The total amount of DNA added to the competent cells is determined by multiplying the volume of the DNA solution by its concentration.
- Volume of DNA: 10 µL
- Concentration of DNA: 10 ng/µL
- Total mass of DNA = 10 µL × 10 ng/µL = 100 ng
2. Calculate the mass of plasmid DNA spread on the plate.
Not all the transformation mixture was used to inoculate the plate. We must determine the fraction of the total volume that was plated and use this to find the corresponding mass of DNA.
- Total volume of cell solution: 500 µL
- Volume plated: 100 µL
- Fraction plated = (Volume plated) / (Total volume) = 100 µL / 500 µL = 0.2
- Mass of DNA plated = Total mass of DNA × Fraction plated
- Mass of DNA plated = 100 ng × 0.2 = 20 ng
To match the standard units for transformation efficiency, this mass must be converted from nanograms (ng) to micrograms (µg).
- Mass in µg = 20 ng / 1000 ng/µg = 0.02 µg
3. Calculate the transformation efficiency.
First, the number of colonies on the plate must be counted. Each colony represents a single successfully transformed bacterial cell. By visual inspection of the image, there are approximately 210 colonies.
Now, the efficiency is calculated by dividing the number of colonies by the mass of DNA plated in micrograms.
- Transformation Efficiency = (Number of colonies) / (Mass of DNA plated in µg)
- Transformation Efficiency = 210 colonies / 0.02 µg = 10,500 colonies/µg
In scientific notation, this is 1.05 x 10⁴ colonies/µg.
Conclusion on Success
The target efficiency for this experiment was 10⁸ colonies/µg. The calculated efficiency of 1.05 x 10⁴ colonies/µg is approximately four orders of magnitude (10,000 times) lower than the target. While the presence of colonies indicates that the transformation process technically worked, it failed to meet the required standard of efficiency. Therefore, in the context of the stated goal, the transformation was not successful. Such a low efficiency would be inadequate for demanding applications like constructing a comprehensive genomic library.
