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Gene copy number by slot-blot hybridization

Candy Tang candysi at hkstar.com
Wed Jan 26 23:05:03 EST 2000


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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