I am trying to do some recombineering to knock out a gene in BL21 DE3 that is interfering with my POI purification. I ordered a plasmid with the lambda red genes in it from addgene which is also temperature sensitive. This came as an agar stab. I streaked multiple plates but was never able to isolate a single colony due to the overgrowth of the cells. I finally just used a glob of colonies. I grew them overnight at 30 degrees with 100 ug/mL Amp. The overnight culture grew just fine and I plasmid prepped it, cut it with two different restriction enzymes and ran it on a gel. The plasmid migrated where I would expect, it looked pretty clean and both RE showed cuts. I have been trying to transform it into a BL21 cells and so far no luck. I made some chemically competent cells and I also used some commercial chemically competent cells and tried heat shocking it, and nothing grew, so I thought because it was temperature sensitive, that wouldn't work. I then made some electrocompetent cells and tried to electroporate and that hasn't worked either.

Electrocompentent protocol:

Started an overnight culture. Next day innoculated 1:100. Grew until OD600 was about 0.7 (I meant to stop them earlier but got caught in a psuedo meeting and didn't get a chance to get to them). Placed on ice for about 15 minutes, spun them down in a 4 degree centrifuge, washed with cold 10% glycerol a few times, and then resuspended in 10% glycerol. Froze some at -80 and tried to electroporate a few right away (I have also tried some of the frozen cells). I tried a few different electorporation settings. Then I immediately put the cells in an epi tube containing 975 uL broth. Shake them for 1 hour at 30 degrees, and plate overnight at 30 degrees.

I did electroporate at pET plasmid as control, and I did get some cells to grow, although they didn't cover the plate as much as I thought. I made got 20 colonies from that.

I'm not sure why this won't work. Is it the plasmid, or the cells?