Selection during Extraction: Plasmid or Genomic DNA? - (Jan/31/2009 )
What's the difference in protocol between the extraction of plasmid or genomic DNA? What determines we get genomic or plasmid in the layer above the protein precipitate?
strength of the denaturation/lysis solution used to break the cells.
plasmid extraction protocol use NaOH and SDS, which has a pH of 14. This causes the DNA to denature into ssDNA. Once the solution is neutralise, the DNA renatures. As plasmids are rings, the DNA is held close together and upon neutralisation, the ssDNA can find each other and reanneal.
Genomic DNA are large molecules so for all practical purposes are linear molecules. Upon denaturation, they lose contact with the partner strand. Upon renaturation, the ssDNA strand can not find their partner strand. Instead they reanneal randomly, producing a very large tangle.
cell lysis solution that extract genomic DNA on the other hand have a pH between 8 to 9. At this pH the DNA doesn't denature. So you can extract the genomic DNA. However, if there are plasmids within the cell, this solution will also extract the plasmid.