Purification of RF DNA from phage prep - Need to get rid of ssDNA contamination (Sep/30/2008 )
I am trying to get pure double stranded DNA from a phage prep I did recently. The plasmid DNA (Replicative Form RF DNA) I get by lysing the phage infected cells contain a small contamination of ssDNA, which is natural since the M13 phage is a ssDNA virus. I had hoped that it would be a simple task to gel purify the RF DNA but unfortunately the yield is extremely poor. (I have tried different silica column kits) Unless you are dealing with linear DNA, which I am not, it seems hard to do gel purification.
I have also tried to do CsCl ultracentrifugation, the ssDNA contamination persists and is clearly visible on a gel. Following at tip I read in these forums I have also tried to to alkaline lysis buffer, followed by neutralization, again to try to precipitate out the ssDNA. That didn't seem to work either, unfortunately.
Does anyone have any suggestions?
maybe s1 nuclease digestion will help rid you of the single stranded dna.
Hydroxyapatite binds dsDNA, but not ssDNA. Check the literature on HA chromatography, or use keywords hydroxyapatite and ssDNA in pubmed.