Transformation is mainly performed in molecular cloning to maintain and propagate, in bacteria, DNA sequences of interest incorporated into plasmids. When analyzing colonies after transformation of cells, you may observe a number of issues, such as few or no transformants, transformants with truncated inserts, etc. This section addresses common problems in bacterial transformation and recommendations on how to solve them.

Possible cause Recommendation
Suboptimal transformation efficiency

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.

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Possible cause Recommendation

Unstable DNA

DNA mutation
  • Mutations may have occurred during plasmid propagation in transformed cells. Pick a sufficient number of colonies for representative screening for sequencing. If all colonies show the same mutation, it may have originated in the original template.

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Possible cause Recommendation
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.
Improper colony selection method
  • Check that the appropriate procedure and host strain are being used for the colony selection method. For instance:
    • Blue/white screening: The host strain must carry the lacZΔM15 genetic marker, and the vector must contain the lacZ gene with the multiple cloning site (MCS).
    • Positive selection: Ensure that the host strain does not carry a genetic marker that confers resistance to the vector’s lethal gene, to ensure that vectors that fail to acquire an insert will cause cell death.
Issues in upstream cloning steps

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Possible cause Recommendation
Antibiotic-resistant strain
Vector plasmid recombined into the host chromosome
  • The vector plasmid may have integrated into the bacterial chromosome, conferring antibiotic resistance. When this occurs, colonies may appear to lack the transforming DNA, since independently replicating plasmids cannot be detected by the usual screening methods (which selectively purify or detect the plasmids and not the chromosomal DNA). Use competent cells with the recA mutation to prevent recombination and allow stable propagation of the transforming plasmid vector.
Satellite colonies
  • Limit the incubation time to <16 hours after plating to avoid antibiotic breakdown around overgrown colonies, which can lead to formation of satellite colonies.
  • For screening, pick well-isolated colonies with no visible satellite colonies.
  • For selection of ampicillin-resistant colonies, consider using the more stable semisynthetic antibiotic carbenicillin.
High number of cells plated
  • Adjust the cell volume and/or dilutions as necessary during plating to reduce and optimize the number of colonies formed. Over-plating of cells could result in degradation of plate antibiotics and growth of cells without transforming DNA.
Cell contamination
  • Use sterile tools and labware, media, and reagents where appropriate or required in the workflow.
  • Make sure spreading rods and/or glass beads are sterile, and cell plating is performed under aseptic conditions.

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Possible cause Recommendation

Large number of cells plated

  • Adjust the cell sample volume and/or dilutions as necessary for plating to reduce and optimize the number of colonies formed.

Long incubation

  • Limit the incubation time to <16 hours after plating to avoid overgrowth.
  • Check the recommended length of time for culture growth, since some strains are designed for accelerated growth.

Improper spreading

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For more troubleshooting tips, please visit our Competent Cells Support Center, review molecular cloning and restriction digestion troubleshooting tips, or contact our technical support team.

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