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  • Troubleshooting Transformation Reactions

    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
    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