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
