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  • Troubleshooting Transformation Reactions

    The following guide can be used for troubleshooting transformation reactions. You may also be interested in reviewing addition tips for Chemical Transformation or Electroporation.

    Problem Cause Solution
    No colonies present
    or no growth
    in liquid culture
    Cells are not viable
    • Transform an uncut plasmid (e.g., pUC19) and calculate the transformation efficiency of the competent cells. If the transformation efficiency is low (<104) re-make the competent cells or consider using commercially available high efficiency competent cells.
    Incorrect antibiotic or antibiotic concentration
    • Confirm antibiotic and antibiotic concentration
    DNA fragment of interest is toxic to the cells
    • Incubate plates at lower temperature (25–30°C).
    • Transformation may need to be carried out using a strain that exerts tighter transcriptional control over the DNA fragment of interest (e.g., NEB-5-alpha F´ Iq Competent E. coli (NEB #C2992))
    If using chemically competent cells, the wrong heat-shock protocol was used
    • Follow the manufacturer’s specific transformation protocol (Note: going above the recommended temperature during the heat shock can result in competent cell death)
    If using electrocompetent cells, PEG is present in the ligation mix
    • Clean up DNA by drop dialysis prior to transformation
    • Try NEB’s ElectroLigase (NEB #M0369)
    If using electrocompetent cells, arcing was observed or no voltage was registered
    • Clean up the DNA prior to the ligation step
    • Tap the cuvette to get rid of any trapped air bubbles
    • Be sure to follow the manufacturer’s specified electroporation parameters
    Construct is too large
    • Select a competent cell strain that can be transformed efficiently with large DNA constructs (≥ 10 kb, we recommend trying NEB 10-beta Competent E. coli (NEB #C3019))
    • For very large constructs (> 10 kb), consider using electroporation
    Construct may be susceptible to recombination
    • Select a Rec A- strain such as NEB 5-alpha (NEB #C2987) or NEB 10-beta Competent E. coli (NEB #C3019)
    The insert comes directly from mammalian or plant DNA and contains methylated cytosines, which are degraded by many E. coli strains
    • Use a strain that is deficient in McrA, McrBC and Mrr, such as NEB 10-beta Competent E. coli
    Too much ligation mixture was used
    • Use < 5 μl of the ligation reaction for the transformation
    Inefficient ligation
    • Make sure that at least one fragment being ligated contains a 5´ phosphate moiety
    • Vary the molar ratio of vector to insert from 1:1 to 1:10
    • Purify the DNA to remove contaminants such as salt and EDTA
    • ATP will degrade after multiple freeze-thaws; repeat the ligation with fresh buffer
    • Heat inactivate or remove the phosphatase prior to ligation
    • Ligation of single base-pair overhangs (most difficult) may benefit from being carried out with Blunt/TA Master Mix (NEB #M0367), Quick Ligation Kit (NEB #M2200) or concentrated T4 DNA Ligase (NEB #M0202)
    • Test the activity of the ligase by carrying out a ligation control with Lambda-HindIII digested DNA
    Inefficient phosphorylation
    • Purify the DNA prior to phosphorylation. Excess salt, phosphate or ammonium ions may inhibit the kinase.
    • If the ends are blunt or 5´ recessed, heat the substrate/buffer mixture for 10 minutes at 70°C. Rapidly chill on ice before adding the ATP and enzyme, then incubate at 37°C.
    • ATP was not added. Supplement the reaction with 1mM ATP, as it is required by T4 Polynucleotide Kinase (NEB #M0201).
    • Alternatively, use 1X T4 DNA Ligase Buffer (contains 1 mM ATP) instead of the 1X T4 PNK Buffer
    Few or no
    transformants
    Inefficient blunting
    • Heat inactivate or remove the restriction enzymes prior to blunting
    • Clean up the PCR fragment prior to blunting
    • Sonicated gDNA should be blunted for at least 30 minutes
    • Do not use > 1 unit of enzyme/μg of DNA
    • Do not incubate for > 15 minutes
    • Do not incubate at temperatures > 12°C (for T4 DNA Polymerase, NEB #M0203) or > 24°C (for Klenow, NEB #M0212)
    • Make sure to add a sufficient amount of dNTPs to the reaction (33 μM each dNTP for DNA Polymerase I, Large (Klenow) Fragment, NEB #M0210 and 100 μM each dNTP for T4 DNA Polymerase, NEB #M0203).
    • When using Mung Bean Nuclease (NEB #M0250), incubate the reaction at room temperature. Do not use > 1 unit of enzyme/μg DNA or incubate the reaction > 30 minutes.
    Inefficient A-Tailing
    • Clean up the PCR prior to A-tailing. High-fidelity enzymes will remove any non-templated nucleotides.
    Restriction enzyme(s) didn’t cleave completely
    • Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence
    • Use the recommended buffer supplied with the restriction enzyme
    • Clean up the DNA to remove any contaminants that may inhibit the enzyme
    • When digesting a PCR fragment, make sure to have at least 6 nucleotides between the recognition site and the end of the DNA molecule