I'm working with the SSGCID plasmid (AVA0421), derived from pET 14b with amp resistance.

I transformed this plasmid into DH5alpha and Rosetta (DE3) Plyss using amp 100 ug/mL, and it worked. Now I'm trying to transform it back into Rosetta (DE3) Plyss and Arctic Express, but I can't!

Method for competent cells: Allow the cell to grow at 0.4, centrifuge, resuspend the pellet in TSS solution, and add glycerol for storage at -86°C.

Transformation (I performed two methods):

i) initial incubation of the sample at 4°C for 20 minutes, heat shock at 46°C for 40 seconds, and incubation in an ice bath for 30 seconds (I've tried this before and it worked).

ii) incubation for 20 minutes on ice and subsequent incubation for 10 minutes at room temperature.

After the shock, 200 uL of LB medium were added to the cells and placed for recovery in agitation at 280 rpm, 27 °C for 1 h

In the positive control, the cells grow well, so I imagine it's a transformation problem.

Any suggestions on what I can do?