Purification of Genomic DNA - (May/18/2005 )
I was purifying genomic DNA using the Wizard Genomic DNA Kit from Promega and usually have no problems with this. One of my samples however had a ton of DNA so after I precipitated my protein I attempted to remove the supernatant containing the DNA into another tube containing isopropanol. Because there was so much DNA, it ended up pulling some protein with it and because I added directly to the isopropanol, I was unable to purify my DNA further. I ended up continuing with the ethanol precipitation and I rehydrated my sample in TE, but tehre is a large cloud of protein in my sample. Can I perform a basic phenol:chloroform extraction on this sample and purify my DNA? If so, does anyone have a good protocol of how I can do this? Also, how long is my DNA stable at 4ºC? Thanks
You don't have to worried for your DNA, because DNA is a very stable is not like RNA.
If, I understand you you want to remove your proteins. Then, try first : add choroform (the same volum of TE), spin 11000xg 5 min, then take the supernatant to another new tube. Add the same volum of Choroform:Phenol( remember that you need different phenol for RNA or DNA, changes the pH of phenol, I don't remember now but later I write you and explain you the kind of phenol that you need for DNA), spin at 11000xg 5 min. Remove the supernantant to a freshly tube and precipitate your DNA with ethanol 75% and AcNa 0.2M o/n at -20ºC. In the morning spin 15 min at 7500 xg and remove supernantat. Redissolve your pellet wit H2O MQ or TE, you can chose. But I prefer water, because TE have EDTA and if you want to make reactions with enzimes sometimes EdTA can interfer in the reaction (because EDTA takes cations that some enzimes need to make the reaction).
I'll hope tihs can help you!!
because you have stacks of DNA I would use larger volumes, maybe resuspend the DNA in 5-20 mls of TE, then perform the phenol chloroform extraction, as you would end up with the same problem of pulling your protein off. The DNA won't go anywhere, just precipitate at the end and resuspend in a lower volume with minimal loss of DNA.