Bacterial Transformation Troubleshooting Guide

To ensure good transformation efficiencies:

• Use best practices when preparing competent cells
• Store prepared cells at –70°C. If possible, store the cells at the bottom or back of the –70°C freezer to minimize temperature fluctuations.
• Avoid freeze-thawing cycles; re-freezing cells lowers transformation efficiencies by about two times
• Thaw on ice
• Avoid vortexing
• Follow the transformation protocol and parameters recommended for the competent cell being used
• To obtain maximum efficiency using chemical transformation, the experimental DNA must be free of phenol, ethanol, proteins, and detergents. Ligation reactions do not need to be extracted or precipitated prior to transformation.
• Consider electroporation over heat shock, for better efficiency with low DNA amount or to generate highest number of clones for library construction
• High salt concentration or bubbles might cause arcing and reduce electroporation efficiency
• Avoid reusing cuvettes; oxidization may have occurred in the metal plates within the cuvette even with a single prior use, and this may affect electroporation efficiency
• Ensure that the properties of the selected cells such as transformation efficiency and genotype are appropriate for the transforming DNA and intended applications, such as methylated and unmethylated DNA, unstable constructs, large plasmids, ssDNA, library construction, etc.
   Refer to the selection guide for competent cells
• Consider a different antibiotic or concentration for clone selection. For plasmids containing both ampicillin- and tetracycline-resistance genes, select transformants on plates containing ampicillin rather than tetracycline, because tetracycline is unstable and can produce toxins that kill transformed 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 selecting a different vector with tighter control elements (e.g., pLATE vectors)
• Consider using a low copy number plasmid as a cloning vehicle
• Grow 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

• Recover the cells in an appropriate medium (e.g., SOC 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, approximately 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

• Ensure that the antibiotic used in the plate corresponds to the vector’s resistance marker
• Verify that the plates have the correct antibiotic concentration

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

Unstable DNA

• For unstable DNA we recommend Stbl2 or Stbl4 for sequences containing direct repeats, tandem repeats, or retroviral sequences. For lentiviral sequences we recommend Stbl3. For inverted repeats, we do not have a strain recommended for this application.
• To help stabilize fragments, collect cells for DNA isolation in the mid to late period of logarithmic growth or early stationary phase, when OD₆₀₀ is between 1 and 2 units
• Pick colonies from fresh plates, <4 days old

• 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.
• Consider using high-fidelity polymerase to reduce chance of accidental mutation during PCR

• If using restriction enzymes (RE) to clone, re-examine fragment sequence to ensure that there are no additional overlapping restriction enzyme recognition sites
• If using seamless cloning method (e.g., Gibson Assembly), re-examine primers used to generate overhangs, you may want to consider using different primers (e.g., with longer overlaps) or re-design fragments
• If cloning extremely long DNA fragment, optimize PCR or consider cloning several smaller fragments using Gibson Assembly

• 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 selecting different vector with tighter control elements (e.g., pLATEvectors)
• Consider using a low copy number plasmid as a cloning vehicle.
• Grow the cells at a lower temperature (30°C or room temperature)

• 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.

• Review troubleshooting tips for upstream steps, such as restriction digestion and cloning

• Check the genotype of the host strain for antibiotic resistance. For example, BL21 Star (DE3) pLysS cells are resistant to chloramphenicol, OmniMAX 2 T1^R cells are resistant to tetracycline, and TOP10 cells are resistant to streptomycin.
• Include a negative control in the transformation step for verification of antibiotic effectiveness. Assuming the correct genotype, untransformed cells should not survive the antibiotic selection.

• 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 standard screening methods, which selectively purify or detect the plasmids but not the chromosomal DNA. Use competent cells with the recA mutation to prevent recombination and allow stable propagation of the transforming plasmid vector.

• 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.

• 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.

• 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

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, such as Mach1, are designed for accelerated growth

• Use appropriate tools and techniques to evenly spread cells on plates

Wrong media

• To increase plasmid yields, grow pUC-based plasmids in TB medium instead of LB medium. TB medium can yield 4–7 times more DNA from pUC-based vectors than LB medium.
• Verify optimal media composition for the strain and application

Improper growth conditions or old colony

• If cells are being grown at 30°C instead of 37°C, incubate for at least 90 min during recovery and incubate the transformed colonies longer
• Use not older than 1 month colony for starting culture
• Ensure good cell culture aeration, use larger flasks with a bigger area to volume ratio. Increasing shaking might be helpful as well.
• Prepare an overnight starter culture and dilute (e.g., 1:100 to 1:1,000) to grow day culture faster (2–3 hours) and harvest a log-phase bacteria