Southern blot - (May/31/2002 )
I'm trying to do a southern blot and I have a trouble, the bands I get are the same bands that appear in the genomic DNA digestion, my probe is specific and it works in northern blot.
I don't really understand the bands you are talking about...after digestion of the genomic dna usually you obtain something like a smear. Is your digestion really efficient?? (ie: 2h @ 37°C, add again enz and then ON @ 37°C for a complete digest).
In my digestion (with XmaI) appears some bands and a smear, but this bands appear ever (I did it 2 times). And are those band I saw in the film. Why?
I've experienced this. The bands are barely discernable in the midst of a smear. Right? Sometime repeat regions in gDNA can generate this phenomenon. So then, when one probes RNA there is much less complexity than with genomic DNA.( This is more true if you are using an end-labeled oligo than a full length or truncated cDNA.) Do you know the size you expect in the genomic DNA blot? Are you going from cDNA to full length gene? How do you know these bands don't contain your sequence of interest? There are many questions you should ask. O.K. the first step is to either vary your SDS and SSC concentrations or increase the hybridization temp to reduce the risk of non-specific binding. You can also wash your present blot more stringently ie higher SDS lower ionic strength higher temperature. Good luck.