Hello everyone!

Recently, I was confused by E.coli electroproration. My goal is to transform the recombinant fragments and pGRB-SgRNA plasmids into competent cells containing pRED-Cas9 by electrotransformation to achieve knockout of related genes.

I made competent cells according to the instructions above of https://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells (100ul 1m IPTG was added to 100mL medium to express Cas9 protein when the cells grew to OD600 was 0.4-0.6). Usually, the concentration of the fragment I added to the tube was about 200 ng, and the concentration of plasmid was about 100 ng. The pulse was released twice by BIO-RAD electrotometer in EcI mode, and then resuscitated at 800ul LB 30 ℃ for 2 hours, then coated on the double-resistant plate. The above process was proceeded on the ice.

However, after transformation, it grows very slowly on the screening plate (usually 18-24 hours, and it often takes 24-48 hours recently), and the final colonies are all wrong by PCR identification.

Researchers are welcomed to discuss the causes of this phenomenon.