Calculating Transformation Efficiency Table 2: Transformation efficiency of E. coli cells transformed with plasmid pUC19 Plate Plasmid DNA Volume Concentration Total DNA Transformation DNA Diluted? of DNA of DNA (ng/μl) used in transformants efficiency added? used transformation (# of colonies) Y or N (M) (ng) LBA n/a n/a LAWN n/a LBA 10 10 ng/μl n/a, LAWN n/a LBAamp n/a LBAlamp 1X (not diluted) 0.5X (diluted by 1/2) 10 20 ng/μl LBAlamp 10 10 ng/μl # To calculate the “total DNA used in transformation,” multiply the volume of DNA used and the concentration of DNA together (the volume units should cancel out)
The Correct Answer and Explanation is:
Completed Table
Exercise C: Calculating transformation efficiency
Table 2 Transformation efficiency of E. coli cells transformed with plasmid pUC19
| Plate | Plasmid DNA added? Y or N | DNA Diluted? | Volume of DNA used (µl) | Concentration of DNA (ng/µl) | Total DNA used in transformation (ng)* | # transformants (# of colonies) | Transformation efficiency |
| LBA – | N | n/a | n/a | n/a | n/a | n/a, LAWN | n/a |
| LBA + | Y | n/a | 10 | 10 ng/ul | 100 | n/a, LAWN | n/a |
| LBA/amp – | N | n/a | n/a | n/a | n/a | 0 | n/a |
| LBA/amp + | Y | 1X (not diluted) | 10 | 20 ng/ul | 200 | ||
| LBA/amp + | Y | 0.5X (diluted by 1/2) | 10 | 10 ng/ul | 100 |
Explanation
Bacterial transformation is a fundamental technique in molecular biology where foreign genetic material, typically a plasmid, is introduced into a bacterium. This experiment aims to transform E. coli with the pUC19 plasmid, which contains a gene for ampicillin resistance, and then to calculate the efficiency of this process. The table outlines the setup and requires calculation of the total DNA used.
Understanding the Controls
Proper controls are essential to validate the experimental results.
- LBA – Plate: This plate contains nutrient agar (LBA) but no ampicillin. Bacteria that did not receive the plasmid were plated here. The resulting “lawn” of growth confirms the initial stock of E. coli was viable and healthy.
- LBA + Plate: This plate also lacks ampicillin but contains bacteria that were subjected to the transformation procedure with plasmid DNA. The expected lawn of growth demonstrates that the transformation process itself (e.g., chemical treatment, heat shock) did not kill the bacteria.
- LBA/amp – Plate: This is a critical negative control. Bacteria that did not receive the plasmid were plated on LBA with ampicillin. The expected result of zero colonies confirms that the ampicillin in the plates is effective at killing untransformed, non-resistant bacteria.
Experimental Plates and Calculations
The “LBA/amp +” plates are the experimental conditions where transformation is expected. Only bacteria that have successfully taken up the pUC19 plasmid will survive and form colonies on the ampicillin-containing medium.
To determine the efficiency, one must first calculate the Total DNA used in transformation. As noted in the table, this is found by multiplying the volume of DNA solution by its concentration:
- For the plate with 1X (undiluted) DNA:
Total DNA = 10 µl × 20 ng/µl = 200 ng - For the plate with 0.5X (diluted) DNA:
Total DNA = 10 µl × 10 ng/µl = 100 ng
These values represent the total mass of plasmid DNA that the bacteria were exposed to in each transformation reaction. To complete the table, one would count the number of colonies on these two plates and use these values to calculate the final transformation efficiency (typically expressed as transformants per microgram of DNA
