* The cells are not competent. Run the controls listed below and obtain new cells if needed.
* The recombinant protein is not well tolerated by E. coli. Try making a fusion with maltose binding protein using the pMAL System (NEB# E8000S). Try another expression system that doesn't involve E. coli.
* The ligated DNA included inverted and tandem repeats selected against by E. coli. Remove the repeat sequence if possible. Try another expression system that doesn't involve E. coli.
* The insert DNA was taken from mammal or plant and contains methylated cytosine which is degraded by many E. coli strains. Use a strain deficient in mcrA, mcrBC and mrr.
* Construct is too large (>10,000 bp) for transformation into chemically competent cells. Use electroporation.