ray at le.ac.uk
Thu Apr 16 10:25:48 EST 1998
In article <6h506u$dns$3 at news.utu.fi>, elebae at utu.fi wrote:
>I have read about DNA depurination in many of the protocols for Northern/
>dot blotting. Does anyone know what is means and what is it done for?
I think that you mean Southern blotting rather than Northern. Depurination
is carried out by soaking the gel in 0.25 M HCl for about 10 min. This
results in random creation of upurinic sites in the still double stranded
DNA. When the gel is then soaked in the denaturing solution (NaOH) the DNA
is made single stranded and the strands are cleaved at apurinic sites.
The reason this is done is to fragment large DNA fragments into smaller ones
which will transfer out of the gel with greater efficiency. Can't think of
a reason why you'd need to do this for a dot blot.
The fragmentation of the DNA is a two-step process. The NaOH step would
hydrolyse all of the RNA in a gel. Hence, depurination is never applied
to Northern blots.
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