Southern Blotting - Possible to sequence from? (Sep/09/2005 )
I'm doing some research to detect how many LWS genes are present in the guppy genome. Our current plan is to digest the genome, run a gel, southern blot, use probes for the gene to detect bands of unique size, then from the gel clone the band out and hybrid screening. Is there a method to sequence from the labelled membrane? My supervisor wants some method where the band is cut from the membrane, washed to get rid of the maker then eluted. I know there will be other bands present due to the multiple cutters we are using, but it is still something of interest.
PCR would be an easy answer, but we are having PCR shuffling problems resulting in recombinant products. Thanks
I have something of a protocol:
"Basically, what you do is run a standard gel electrophoresis and then denature the DNA with sodium hydroxide about 0.25-0.75%. This splices the double strands from the single strands - you only want the single stands, I think.
Then you can neutralize the base with an acid solution at this point in order to remove purines, but I'm not sure that's what you need to do - that's what I had to do in my work anyway.
At any rate, neutralize everything at this point.
Transfer your specimen to a nitrocellulouse membrane, or something else you know the properties of. Then you hit it with a UV light to crosslink the DNA - you could probably also just bake it at around 70C for an hour or two.
Now you hybridize the probe DNA. So you've still got a double stranded sample to use as a probe, right. Depending on what you are after add the apprpriate splicer: maybe dnase, dNTPs, whatever is required - but you need a marker, I think one is 32pATP."
Anybody have any better suggestions?
I think you will find it difficult to get the DNA off the membrane intact. Even if you were able to get the DNA off you will then have the problem that many other sequences other than desired will be present. What exactly do you want to know? Just copy number or do you want to know the sequence as well?
DNA sequencing services
Didn't see this reply...
We want the copy number and the sequences. Found another way to do it, thanks for the offer to help.