Cloning Basics Support - Troubleshooting

Some possible causes and remedies are:

• Ligase function is poor. Check the age of the ligase and function of the buffer.
• Competent cells are not transforming. Test the efficiency of the cells with a control supercoiled vector, such as puc19.
• Both molecules were de-phosphorylated.
• Inhibition of ligation by restriction enzymes and residual buffer. Try transformation of uncut vector, clean up restriction with phenol, or carry out PCR cleanup/gel extraction before ligation.
• Incorrect antibiotic selection used. Check the plasmid and plates and make sure concentration of antibiotic used is correct.

If nothing above applies, low to no colonies may be due to instability of the insert DNA in your competent cells. In this case, E. coli strains such as Stbl2™, Stbl3™, or Stbl4™ have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.

This may be caused by the instability of the insert DNA in TOP10 E. coli. In this case, E. coli strains such as Stbl2™ or Stbl4™ have been shown to support the propagation of DNA with direct repeats, retroviral sequences, and DNA with high GC content better than other strains.

One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium, but is much stronger in faster growth conditions like liquid medium. Suggestions:

1. Use TOP10F’ or any other strain with the LacIq repressor.
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27–30 degrees C and grow the culture longer.
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage-resistant like DH5α-T1R.

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

• After transforming TOP10 or DH5α cells, incubate at 25-30 degrees C instead of 37 degrees C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
• Try using TOP10F’ cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
• Try using Stbl2 cells for the transformation.

These small colonies are most likely caused by degradation of the ampicillin. The colonies are untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:

1. Plate cells at a lower density.
2. Use fresh LB+Amp plates or replace ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. β-lactamase, the enzyme produced from the ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.