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
|
