|Suboptimal transformation efficiency|
- To ensure good transformation efficiencies, prepare competent cells diligently, store them at –70°C with minimal freeze-thawing, and handle them appropriately (e.g., thaw on ice, avoid vortexing).
- Follow the transformation protocol and parameters (heat shock or electroporation) recommended for the selected competent cells.
- Consider electroporation over heat shock, for better efficiency with challenging DNA (e.g., library construction).
- Make sure the properties of the selected cells (e.g., transformation efficiency, genotype) are appropriate for the transforming DNA and intended applications, such as methylated and unmethylated DNA, unstable constructs, large plasmids, ssDNA, library construction, etc. (Also refer to the selection guide for competent cells)
- Include a positive control in the transformation to verify competence and transformation efficiency of the prepared cells.
Suboptimal quality and/or quantity of transforming DNA
- For transformation with ligated DNA, ligase and other reaction components carried over can reduce transformation efficiency. For heat shock, do not use more than 5 µL of ligation mixture for 50 µL of competent cells. For electroporation, the DNA should be purified from the ligation reaction prior to transformation.
- If polyethylene glycol (PEG) is used in the ligation reaction, avoid heat-inactivating the ligase after the reaction. Using heat-inactivated ligated DNA with PEG, without purification, can reduce the efficiency of chemical transformation.
- To achieve maximum transformation efficiency, use appropriate (avoid excessive) amounts of DNA. For example, 1–10 ng of DNA per 50–100 μL of chemically competent cells and 1–50 ng (in ~1 μL) DNA per 20–25 μL of electrocompetent cells generally work well.
Cloned DNA or protein that is toxic to the cells
- Use a strain specially designed for gene expression, with a tightly regulated inducible promoter to ensure minimal basal expression and high expression upon induction.
- Consider using a low copy number plasmid as a cloning vehicle.
- Grow the cells at a lower temperature (30°C or room temperature) to mitigate toxicity.
Incorrect strain used to propagate a vector carrying a lethal gene for selection
- For maintenance or propagation of an empty cloning vector carrying a lethal gene (e.g., ccdB), ensure that the strain is resistant to the toxic gene product.
|Insufficient number of cells plated|
- Recover the cells in an appropriate medium (e.g., S.O.C. medium) after transformation, and allow sufficient time for growth (e.g., 1 hour) before plating.
- Adjust the cell sample volume and/or dilutions as necessary for plating to obtain numbers of colonies within a desirable range (e.g., 30 to 300 colonies per plate).
Suboptimal growth time and temperature
- Allow sufficient time for the cells to grow during recovery and plating (e.g., 1 hour and 16 hours, respectively). Incubation below 37°C may require more time for adequate growth.
- Ensure the incubator is at the correct temperature for growth of the competent cells used. Prewarming the medium and plates may help with cell growth.
Incorrect antibiotic or concentration in plates
Improper use of cell-spreading tools
- If reusable cell spreaders or glass beads are sterilized by ethanol, flame, or autoclaving prior to the plating step, make sure they are free of ethanol or properly cooled before spreading cells.