I am supposed to use Stbl3 E.coli strain for one of pLenti6.3 vectors, due to lack of it I transformed into DH5a only. I got 4 colonies and felt happy and proceeded to isolate plasmid DNA and I made glycerol stocks as well.
I got very good plasmid and I confirmed with RD. Now I want to isolate again the same plasmid DNA using glycerol stock after inoculating freshly. This time I am getting plasmid DNA in the nicked form only not other two forms that are also not getting digested with any enzyme which were used earlier for confirmation. What should I do?
The approach I would follow is to transform again with DH5a cells but do all growth steps at room temperature as opposed to 37 degrees. By growing in a warmer temperature you're exerting selective pressure on the population of transformed cells, and more likely the plasmid is suffering gene rearrangements especially if LTR elements are present. Remember that Long Terminal Repeats is the mechanism used by the virus for gene integration into the genome and thus is the same mechanism of excision from the genome (or the plasmid in this case). Basically, you get deletions. Hope it helps.
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