DNA isolation by PCI method - (Dec/19/2005 )
I have been trying to isolate genomic DNA from murine tissue using PCI method. I have to use this genomic DNA for further quantitative study (Real time PCR). I am really disappointed by myself, for I feel I cannot get this basic technique to work.
I have used several different modification of Manniatis but so far I have not been successful in getting a high molecular band (~3000 kb), that I got when I used a DNA isolation kit from Gentra. I get a smear which is between 600 to 100 bp. I think that I am purifying RNA or my DNA is getting sheared. I am using the right PCI solution (pH 7-8). I might be doing something wrong which I am not able to pick up. I am not sure what is going on?
a dark smear that migrates at 300pb around is RNA
So what am I doing wrong that I am not not getting genomic DNA?
Are you vortexing in your method? This is going to shear your genomic DNA. If so, try gently inverting and be very careful when resuspending.