Transformation efficiency is a measure of success in molecular biology, quantified by the number of bacterial colonies formed for every microgram of plasmid DNA used. A higher efficiency indicates a more successful experiment, which is important when working with rare DNA fragments or constructing extensive gene libraries. Achieving a high yield of transformed cells depends on the quality of the bacterial cells, the purity of the DNA, and the precision of the procedure. By optimizing each stage of the process, from cell preparation to final plating, it is possible to improve the number of successfully transformed colonies.
Preparing High-Quality Competent Cells
Preparing competent cells—bacteria treated to accept foreign DNA—is a foundational step. This state of competency is not natural for most lab strains of E. coli and must be induced through either chemical treatment or electroporation. Chemical competency is achieved by treating cells with a calcium chloride solution to permeabilize the membrane. For electroporation, cells are washed with a non-ionic solution like glycerol to remove salts before an electric pulse is applied.
Harvesting cells at the correct growth stage is important for preparing competent cells. Bacteria should be collected during the mid-logarithmic growth phase, a period of rapid division identified by measuring the optical density (OD600). While optimal OD600 values vary between protocols, this measurement is a guide for peak competency. Growing cells at lower temperatures, such as 18 to 20°C, may also improve their ability to take up DNA.
Laboratories can prepare their own competent cells or purchase them commercially. Commercial cells offer convenience and guaranteed efficiency at a higher cost, while in-house preparations are more cost-effective and allow for control over the strain and method. Different E. coli strains like DH5α, TOP10, or BL21 are suited for specific applications, such as plasmid amplification or protein expression, and have varying inherent transformation efficiencies.
Vector DNA and Ligation Considerations
The quality and form of the introduced DNA are highly influential. Plasmid DNA must be pure and free from contaminants like salts, ethanol, or detergents that can inhibit transformation. The physical form of the plasmid also dictates how easily it enters the cell. Supercoiled plasmid DNA, the compact native form, transforms much more efficiently than nicked or linearized DNA.
The amount of DNA added to competent cells requires optimization, as adding too much can be counterproductive. Adding between 1 and 10 nanograms of plasmid DNA in a small volume yields the best results. Exceeding this amount can saturate the cells or introduce excess buffer, interfering with the chemical balance needed for transformation. Plasmid size also matters, as efficiency decreases with larger DNA molecules.
When transforming cells with a ligation product, where new DNA is inserted into a plasmid, other factors are considered. The molar ratio of the vector to the insert during ligation must be optimized to ensure most vectors are modified. After the reaction, it is beneficial to purify the DNA to remove the ligase enzyme and its buffer. These components can interfere with transformation and reduce efficiency.
Critical Parameters of the Transformation Procedure
Introducing DNA into competent cells requires precision. The heat shock method for chemical transformations involves a sequence of timed temperature changes. Competent cells and plasmid DNA are first incubated on ice for up to 30 minutes, allowing the DNA to adsorb to the bacteria. This is followed by a brief heat pulse at 42°C for 30 to 45 seconds, which creates a thermal imbalance that helps move the DNA across the cell membrane.
Following the heat pulse, the mixture is returned to ice for several minutes to stabilize the cell membranes. Any deviation from the optimal time or temperature during the heat shock can be harmful. A pulse that is too long or hot will kill the cells, while one that is too short or cool will be insufficient for DNA uptake. These parameters may require optimization depending on the bacterial strain and preparation method.
Electroporation uses a high-voltage electrical pulse to create temporary pores in the cell membrane, allowing DNA to enter. This method requires pre-chilled electroporation cuvettes to prevent heat damage to the cells during the pulse. The instrument’s settings, such as voltage and pulse duration, are factors for success. A higher field strength, like 12.5 kV/cm, can yield higher efficiencies, but over-pulsing the cells leads to cell death and a reduction in viable transformants.
Optimizing Post-Transformation Recovery and Plating
After the transformation pulse, cells are fragile and require a recovery period known as outgrowth. This step is facilitated by adding a rich, non-selective liquid medium, such as SOC broth. This broth provides nutrients for the cells to repair their membranes and begin expressing the antibiotic resistance gene from the plasmid. This expression is necessary for the cells to survive the subsequent selection step.
The outgrowth procedure involves incubating the cells in recovery medium for about one hour at 37°C with gentle shaking for aeration. This period allows the antibiotic resistance protein to accumulate within the cells. Without this recovery, many cells that successfully took up the plasmid might be killed when plated on antibiotic media because they have not yet produced the protective protein.
The final step is plating the recovered cell culture onto agar plates containing the selective antibiotic. Using pre-warmed plates can help the liquid culture absorb more quickly and may improve efficiency. Plating too large a volume can result in a lawn of colonies instead of distinct ones. Plating between 50 and 100 microliters is common practice that allows for the development of countable colonies.