Hi everybody
I am doing southern blot to confirm the presence of transgene in plant .I have some dought regarding on this topic. for doing southern I am using alkphos kit.
!)Can we use more conc of probe than what is there in instruction manual.
2)how can we predict nonspecific binding of probe.
3)is there any method to calculate the hybridization temp
Hi,
1.) We normally use just half of the probe written in the manual and get good results with this.
2.) This depends on how you designed your probe. You should have it in a region without repetetive sequences and I made the experience that larger probes (700-1000bp) work better than smaller ones for my region. This must of course not always be the case.
3.) we incubate all probes at 65°C over night.
Hope this helps!
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