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Gene copy number by slot-blot hybridization

Lluís Armengol llarmengol at iro.es
Thu Jan 27 02:02:14 EST 2000


    Hi,

    How do you fix the DNA on the membrane? Sometimes, just drying the membrane
would be enough; but if you want a better fixation you may try to use UV
irradiation or heat fixation (placing the membrane in an incubator at 80ºC for
1.5-2 hours would do both a partial denaturation of the DNA and fixation to the
membrane).

    regards, Lluís

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??
>
> Regards,
> Lum
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