I too faced the same situation

Is your control only plasmid DNA containing your gene and nptII? Sometimes the problem with Southern blot is the amount of genomic DNA used, which could be not enough for the the sensitivity of the method. For a big genome such as maize's, I have seen they use 15-30 micrograms, for Arabidopsis 2 micrograms is usually enough. When performing Southern blots, I include a control combining genomic DNA (from non-transformed plant) with plasmid, in such a proportion that the gene of interest is in one copy per diploid genome (something you would expect from your transformed plants). That way you can really compare presence / intensity of bands between your control and putative transgenic plants.

Regarding your plants, if you got positive results through PCR, and no amplification form control plants, you can be sure your plants are transformed, provided you have included appropriate controls. If you really want to be on the safe side, you could run PCR and perform Southern blot on those bands, just to be sure you are not having unspecific amplification. However, if you are including a non-transformed plant as control, unspecific amplification can be ruled out that way.

Hope it helps! :)

Dear Dr,

I have the same problem with southern blot that you mentioned in your question. I got good band in control whereas in mutants there are faint or nothing. I am waiting your reply please.

Regards

Ayet Al-Laaeiby; I overcame this problem by using large amount of plant DNA. I used 50 ug of DNA for restriction digestion and loaded all in the gel( The gel was thick with large comb).

Thanks Gustavo for your suggestions.