Hi everyone,
I tried making my own RNAi clone for the first time (by cloning a ~800 bp piece of my gene of interest into L4440 and transforming into HT115 E. coli). The RNAi clone grew fine in LB/Ampicillin when I grew the overnight culture, BUT when I put the bacteria on RNAi plates containing IPTG and selection antibiotics the bacteria died. Is it possible that the dsRNA expression somehow killed the bacteria? If so, what can I do to present this - i.e. how should I redesign my RNAi cloning? I already checked to make sure I hadn’t disrupted the antibiotic resistance genes during my cloning, and in fact I didn’t (I only used rrestriction sites in the MCS). I have used many Julie Ahringer library RNAi clones without ever experiencing this problem, and in fact my control RNAi and other RNAi clones grew fine on my RNAi plates this time too!
Thank you very much!