I have successfully made a construct for knock-out by overlapping extension PCR with Kanamycin selection marker (RR-Kan-RF, FF=312bp, RF=313bp, Kan=937bp) for Acinetobacter baumannii (Gram -ve). I had used Taq pol for construction, still got the construct at 218 ng/microliter purified using elution buffer in the PCR purification Kit. I used 1 microgram DNA (linear construct) for electroporation at 1800 mV. Cells are electro-competent as I can get transformants with a control plasmid. Please suggest.