Gene copy number by slot-blot hybridization

Colin Rasmussen colin at pombe.usask.ca
Thu Jan 27 11:14:03 EST 2000



Candy Tang wrote:

> Hi,
>
> > >       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??
> >
> > Are you denaturing your genomic DNA before you apply it to the filter?
> >
>
>        Yes, by adding NaOH to final conc. of 0.4N and boil for 16 minutes.
> However, after the slot blot process, I notice something stick on the
> membrane. This is only observed in slot with high amount of DNA (e.g. 35ug) Is
> that the DNA..?? And how should I fix the DNA on the membrane??

Why would you add that amount of NaOH AND boil.  Sounds pretty harsh to me.  I'd
simply boil it for 10 minutes, cool in ice, and then apply to the blotter.

Colin






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