I have inserted a plasmid (using a pET28a+ backbone, expressing the lac0 operator) into chemically competent BL-21 DE3 cells. I had selected for kanamycin and plated. I then picked a colony and grew up a 250 mL culture in SOC media following the NEB protocol to create electrocompetent cells. I noticed, however, when attempting to electroporate in mRNA, which expresses CAM resistance and is replicated by a replicase encoded on the pET28a+ plasmid, that none of my cells survived on CAM. I believe this is because the replicase (target protein) is not being expressed due to lac suppression. I would like to remake the electrocompetent cells to be preloaded with the replicase, but don't know when IPTG should be added as its optimal OD600 aligns with that which is desired for electrocompetency. Any advice would be much appreciated! Thank you!
Just an idea: why not to grow them in soc medium containing lactose after electroporation?
Miguel Fernández-Niño I had thought about doing this as well and will be running the experiment today. The timing, however, as to when I should plate is uncertain due to the delayed growth of bacteria in the presence of IPTG. Thanks for your advice!
Good day,
The first thing you have to do, once you have your construct ready, either if you are using electroporation, osmotic or temperature stock, etc., to perform transformation, is to prepare competent cells, then carry on with the transformation, use 25 mL of SOC to grow them, use a petri dish to perform a massive cell culture, grow overnight. Once you confirm the positive transformation, add the above menciones 25 mL of SOC to a fernbach flask containing the media of your choice and grow them. IPTG should be added to your, already competent and transformers, bacterial cell once they have reached a minimun OD600 of 0.6, although some people prefer to add it on late logaritmic phase (~1). I'd recommend you to go with classical density and only move from there if it is absolutely neccesary.
Best regards
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