plasmid + sepharose - how do this work? (Jun/30/2010 )
Hi to all,
I had normally made maxipreps using the super easy kits protocols (i.e. Qyagen, Invitrogen...) but i have arrived to a lab were they make the maxipreps in a old way using home made solutions ans sepharose columns.
Briefly, the procedure involves an alkaline lisis, clearing, RNAse A incubation, Phenol-cloroform extraction and passing the resulting suppernatant to the sepahrose column for recovering several fractions, some of them containing the plasmid.
I did it once but got really few plasmid!!!
My question is, if somebody still do it in this way, which is the function of the sepharose column? it it really neccesary after phenol cloroform extraction?
the sepharose column is to separate the molecules by size. in your case, separating plasmid from genomic dna.