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Probes design for southern-blot


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#1 Xavier

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Posted 12 April 2010 - 06:33 PM

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

#2 bob1

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Posted 12 April 2010 - 07:19 PM

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?

#3 Xavier

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Posted 12 April 2010 - 07:37 PM

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?


Thanks bob1 for all this answer.
For the fact to use several probe I thinking to use a probe for detect the EGFP DNA (because my transgene construct is like that: gene of interest-IRES-EGFP) and another one to detect the intronic region of my native gene. What do you think about it?

thanks again,

Xavier

#4 bob1

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Posted 13 April 2010 - 04:12 PM

Your plan sounds fine to me




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