Hey, i'm doing GISH(Genomic in situ hybridization) with plant material,
and I'm struggling with indistinct signals. Proper ratio seem to be different from species to species..
What I'm wondering is, is there a big difference between using herring sperm and plant genome as a blocking DNA in GISH?
Since there are so many variable, so even if I change blocking DNA, I don't know whether problem is caused from blocking DNA or other...
Of course using own plant genome as a blocking DNA should be better, but I'm wondering if there is no difference between using herring sperm and plant genome, then I can eliminate one variable.. help me!
Hi Somin,
If you are studying interspecific hybridization, depending on how related the progenitors of the hybrid, the probe/blocking DNA ratio must be enough to prevent the labeling of chromosomes of both genomes at the same time. Please see the attached paper p2955 and p2957 (See attached paper, yellow highlight).
Ex. "It was necessary to use 100X concentrated blocking DNA in hybrids of Lycopersicon esculentum and L. peruvianum to distinguish the genomes (Parokonny et al., 1997), whereas in hybrids of Dendranthema nankingense and Tanacetum vulgare, both genomes were identified without the need of a blocking DNA " (p2955).
You meant as the use of herring sperm or salmon sperm DNA in Southern or Northern hybridization for blocking agent? The use of these DNA in Southern/ Northern analysis is to reduce the non-specific bindings and reduce background of a blot.
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