Master plate of DH5a is not working now, but I have that plasmid in rosetta that contains its own plasmid also. So how can I isolate and purify my own one?
Hi. I haven't worked with the Rosetta strain myself, so I know nothing about their native plasmids, but if you know the size of your own plasmid, I guess you can simply do a plasmid-prep followed by gel extraction. Hope this is useful :)
Hi Vaishnavi,
Just plate your cells on LB plates supplemented with Ampicillin antibiotic (final concentration of 100ug/mL). Your pRSET-B vector has an Ampicillin resistant gene where as the Rosetta and its other derivative strains has chloramphenicol-resistant gene in its plasmid. Only cells harboring the ampicillin resistant gene (in this case your pRSET vector) will grow on the agar plate. You can then mini-prep to extract the plasmid out of the cells.
Good Luck!!
@ Saranpal Chhabra,
That won't work. The pACYC derivatives harbored by the Rosetta strains are based on the p15A replicon, which is compatible with the replicon of the pRSET series. Hence minipreps from your Rosetta transformants will have both plasmids (p15A-derived plasmids are not lost that easily, even in the absence of selective pressure).
A better solution is to just retransform the minipreps from the Rosetta transformants into your strain of choice. Since the odds of co-transformation are lower, it is easy to get clones containing only the pRSET-B plasmid.
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