a. The cells are not viable or competent. Run the controls listed below and obtain new cells if needed. b. 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. c. 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. Note: Use the same DNA concentration for each control, typically 0.1-1 ng per transformation. i. Uncut vector to check cell viability and test the antibiotic resistance of the plasmid. Plate on media and media plus antibiotic. ii.Vector cut and not ligated: to check for uncut vector, gel purification may be required to remove the last 0.1% of uncut vector. iii. Cut and religated plasmid (no insert) to check ligase activity and DNA end integrity. Should be 60-70% of the value obtained for uncut vector. iv. Cut, dephosphorylated and religated plasmid to check background due to incomplete phosphatase treatment. Should be < 3% of the value obtained for uncut vector.