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Gene copy number by slot-blot hybridization

Colin Rasmussen colin at pombe.usask.ca
Thu Jan 27 11:15:27 EST 2000



David Barrass wrote:

> On Thu, 27 Jan 2000 00:20:19 +0800, lumdicks <lumdicks at netvigator.com>
> wrote:
>
> >       I'm planning to use slot blot hybridization to calculate the gene
> > copy number in the genome. However, my slot blot give a very weak signal
> > even 50ug of genomic DNA was applied. Is there any possible casue for
> > this..?? I am using DIG labelling and chemiluminecence detection. The
> > system works fine for southern blot (using the same probe and genomic
> > DNA), which can detect a single copy gene using 5 - 10ug of genomic DNA.
> > So, I don't know what's wrong with the slot blot. Can anyone give any
> > suggestion??
>
> I battled with the same problem trying to determine copy number in
> transgenic animals.
>
> Let me guess are you looking at mammalian genome?   If so I suspect
> that this approach will not work :-=(.  There is too much negative DNA
> in the way with large genomes.; the probe simply cannot find the
> target.  I eventually concluded that Southerns were the only way until
> I could persuade someone to buy me a Quantitative PCR machine.

Not true.  I've done it using tissue culture cells transformed with a
plasmid, and I wanted to determine the copy number of the plasmid.  One thing
that might also help is to RE digest the genomic DNA prior to denaturation
and blotting.

Colin





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