I was working on plasmid isolation from an organism but i didn't got plasmid via plasmid isolation kit. However, later on i extracted the gDNA from that portion of the given organism, luckily i got the plasmid extracted with the genomic DNA. can anybody please suggest me any protocol or any paper, how to isolate it from the DNA?
I can think of two options:
1- retransform the mix in a proper host then extract the plasmid properly
2-digest the mixture with multiple enzymes that do not cut in the plasmid (to clear the genomic DNA smearing) then run the mix on a gel and cut out your plasmid.
digest the mixture with multiple enzymes that do not cut in the plasmid (to clear the genomic DNA smearing) then run the mix on a gel and cut out your plasmid.
Should have just used a plasmid prep kit on the organism you have extracted the DNA from. I would just start again and do that as will save you time and effort.
There are a couple of tricks that might work here:
(1) Run uncut DNA on an agarose gel and cut out the plasmid bands (there should be three: linear, open circular, closed circular.
(2) Denature with 0.1 N NaOH and then neutralise with 3M Sodium acetate. This will cause the genomic DNA to clump together by semi-random basepairing, while closed circular plasmid DNA will renature correctly and remain in solution. This is essentially the same as the classic Birnboim-Doly plasmid prep except that the material is DNA instead of bacterial cells.
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