Probes design for southern-blot - need more information for probes design for southern-blot (Apr/12/2010 )

Hello everybody,

I try to design a probe for my southern but I have a problem.
Indeed I try to detect the number of copy of my transgene and my native gene (wich are basically the same but my transgene have only the cDNA sequence).
My problem is than the exons of my gene of interest is very short and the gene probes I could do for this gene is lower than 100pb.
So can I use a gene probe lower than 100pb for my southern? I wanted to know if it was a problem because I read that the majority of the genes probes were made between 500 pb and 1000 pb.
Or maybe should I do oligonucleotides probes?
An oligonucleotides probes works for DIG-labeling?
Also I want to know if I could digest my gDNA by several enzymes?
Or Can I use several probes in the same southern? one probe to detect my transgene and an another one to detect my native gene?And Can I quantify my transgene if I use this way?

thanks a lot to answer at all my questions, I really need help....

Xavier

Oligonucleotides should work if designed appropriately... after all PCR works off 20 bp oligos for specific amplification. DIG labelling seems to be the way to go, I have used it for Southerns and it works very well. I was making probes by PCR, so there was a lot of DIG being incorporated, making detection easy.

You could digest your DNA by several enzymes, but it is best to chose just one, so that the DNA separates into a nice range of fragments, that are easily separated by an agarose gel.

You can do sequentially detect genes on a Southern - if you try to use more than one probe at a time, how will you know which band is which?

bob1 on Apr 12 2010, 10:19 PM said:

Your plan sounds fine to me