southern blot probe design - (Dec/21/2006 )
I am new here
May someone can help me
I would like to do a southern blot to screen for alternative splicing variants of 5 genes in different cDNA libraries
I am thinking to use radiactivity labeling.
I have each gene cDNA cloned into vector that i can use as positive control and to generate probes.
My question is about the probe design: I have different options and I'd like to know which one is the more sensitive :
- extract the cDNA using restriction enzyme. The longest is about 1.8 kb.
- extract only the conserved domain between all the variants (that is around 300 pb long) using restriction enzyme or PCR it
- design a short specific oligo (40 pb)
Thanks in advance for your help
Ordinarily, longer is better for Southern blot probes. However, given what you're trying to do, I think your PCR strategy makes sense. And since you're screening a cDNA library, there should be plenty of sensitivity in a probe of this length.
The oligo approach ought to work also, and can be done very quickly (oligo hybs and washes are fast).
For this purpose I'd avoid using the full-length cDNA as a probe, since this will give you much better signal from clones containing that specific splice form and not from the others.
However... maybe I misunderstood your question... you're not talking about designing a *single* probe that will detect all five genes, are you? That would be a bad idea I think. Specific probe for each gene would be much, much better.
Member of The Science Advisory Board
Thanks Alveron for your answer .
PS: You understood right i will use a specific probe for each gene.