I have purchased this plasmid from addgene:
https://www.addgene.org/70719/
From the agar stab, I have been able to grow the plasmid on LB-ampicillin plates (100mg/L) and it grows great. I isolated the plasmid using a miniprep kit, and it is exactly where it should be on a gel.
Then I tried to transform the plasmid into BL21(DE3) competent cells (NEB). No luck.
I have been troubleshooting this for months. I use pUC19 as a positive transformation control, and it always works really efficiently. I have tried changing DNA concentration, volume, heat shock time, recovery time, everything I can think of. I cannot get this plasmid into cells. Please help!
What protocole do you use for constructing the plasmid?
if yoi use restriction-ligation, you need to make sure that you dont use any restriction enzyme that cut " outside" the polylinker site. For example, if you use Scal, Pvul or Bsal enzyme, the plasmid will loose the Ampicillin resistance activity.
To check the constructed plasmid it is better first to perform transformation in DH5a, check and multiply the plasmid. BL21 are better for producing proteins
Have you tried transforming your plasmid preps into a different strain of E. coli just to double check that the plasmid preps are good and that there's not something weird going on with that?
If it's not that then maybe try adding 10 mM glucose to the recovery broth and plates, in case it's an issue of leaky expression.
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