Hi,

I am trying to transform 10 Kbp plasmid vector ( my gene of interest is around 5 kbp) in the Pectobacterium. Usually, we can't make pectobacterium chemical competent, so we make them electrocompetent. The plasmid doesn't contain OriT, so I can't transform it by conjugation. I used 400 ng plasmid DNA ( miniprep with Qiaquick kit) for the transformation. I found only 1 colony in the plate which is very weird because I got a plate with colonies when I used same competent cell to transform the same empty vector ( 5 kbp, this one don't have my gene of interest). I added 1 ml of LB immediately after electroporation and incubate the cells at 25 °C for 2.5 hr.Then pellet by centrifugation at 5000 rpm for 10 min and pour or most supernatant. Resuspend the pellet in the little supernatant (~100 ml) and plate that into km antibiotic selection. How I can improve my transformation.

Thanks in advance.

Cheers