Hello everyone,
I had some plasmids synthesized by TWIST. I transformed them into homemade chemically competent NEB-stable. During the outgrowth, I accidentally grew them at 37C instead of 30C, as recommended by NEB. I did the final plating/incubation at 30C, as recommended.
I don't believe my inserts are particularly unstable; they're just E. coli K-12 genes under the T7lacI promoter. I wanted them in NEB-stable due to the Laciq mutation that NEB-stable has to make lacI even stronger at repressing.
My question is, would the 37C outgrowth have that big of an impact on transformation efficiency?
You should be fine. "Stable" E. coli cloning strains (NEB-stable, the Stbl strains, etc) are primarily meant to prevent recombination in plamids destined for mammalian cells like lentiviral vectors, which have long repetitive elements. It sounds like that's the primary concern for recombination at elevated temperatures. While bacterial genomes do have problematic repetitive regions, they're uncommonly things you would clone into a plasmid (with the exception of say, some transposon vectors).
Also, as far as I can tell, the NEB-Stable strain does not even contain T7 RNA Polymerase required to even express genes driven by the T7 promoter, so your genes should be silent and not confer any selection advantage to mutants even for toxic proteins, barring anything like unexpected like cryptic expression from an unexpected promoter upstream in the plasmid, of course.
Sequence verification is important for any cloning project though.
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