I am trying to detect the transgene copy number in Arabidopsis thaliana ecotype col 0 with Alkphos labeling and detection kit. As per the manufacturer's instructions I am using 20ng probe per ml of the hybridization solution and I have used about 1.5 µg of genomic DNA. I am not getting any signal with native as well as the transgene probes. Could you please suggest to me the amount of genomic DNA and probe I should be using to get a decent signal. Would ethidium bromide staining of the gel post-run have an effect on the transfer of the DNA onto the blot?
Hi Anil,
For standard Southern, the EtBr-stained gel should not have caused a problem for transferring the DNA onto the blot. I have always used EtBr to stain my gel (for taking a picture and document the distance of each DNA markers) before transferred the DNA onto a blot. For me, it has no problem. Good luck!
Hi anil,
How are you estimating the probe concentration.
You can increase the probe concentration and gDNA concentration and check .
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