homolog detection using Southern - (Mar/01/2010 )
I am trying to use a Southern to detect the number of homologs. I suspect there are three but need to test. If I use a dot blot to determine the concentration of gDNA to run out on the gel, will that number actually be correct? If, for example, my probe shows a nice signal with 10ug dotted, won't the signal be much weaker when the DNA is all separated out on the gel? Is that linear? Can I just scale up?
Thanks for the help.
If you are using all three probes in the same hybridisation, the the signal for each should be approximately a third of what you see on the dot blot, assuming equal binding and labeling of the probes.
I would try to dot blot with each of the probes separately to see the signal strength of each, and then use the weakest as the basis for loading.