I was wondering if we could skip one step in cloning process from sequencing vector to expression vector? Meaning that start cloning the gene directly into expression vector such as pET 28b and do sequencing; checking the sequence go ahead for expression using DE3 cells.
Please share your thoughts
Thanks,
Mah
I recommend you to start with a cloning vector, such as pGEM-t easy. In that way, you can select the positive clones in a Blue/White Screening on indicator plates. If you start the cloning with pET you don't know a positive clone till you do the sequencing.
You certainly can sequence pET plasmid DNA and it is fine for storage as well. However BL21 is not a great host for making high quality DNA because it still expresses the periplasmic nuclease endA. My lab usually propagated clones in pET vectors by using a non-expressing host (DH5 or anything similar) and sequenced from that prep. Then we would transform an expression host with the confirmed plasmid (like BL21 (DE3).
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