Download citation...
Asked 3rd Oct, 2012
  • Central Proteina Prima Company of Jakarta, Indonesia

How to design probe specific hybridize with a specific targeted gene in Southern blot method?

I use DIG-11-dUTP label probe with specific targeted gene to detect in host genome. Host genome have a huge sequence that to date no information in data base. I cut the genomic DNA by restriction enzymes but after southern blot process my specific gene in the host genome does not show the signal band. I use chemilluminence (SDP star) substrate for alkaline phospatase, I do not use radioactive label 32P. Could anyone give me more ideas with this case?
17th Oct, 2012
Mohan Harikrishnan
Louisiana State University
Initially you can try washing the blot with low stringency conditions and check for signals. Since plasmid DNA are small and intact they will produce good signal but its not the same with genomic DNA.
You can try the following steps. It may look silly but sometimes it will work better
1. Minimise the thickness of your agarose gel
2. Also you can try with SSPE buffer for transfer instead of SSC
2. UV-crosslink your blotting membrane before hybridisation for 2 mins
3. Optimise your DNA-DNA hybridisation temperature
4. Try low stringency washing.