half-size fragments after QiaQuick purification?

MaikelG m.giesbers at NOBULLSHITncmls.ru.nl
Tue Apr 26 02:04:15 EST 2005


Hi 

Indeed before the purification the size was ok, and I cut the right size 
of band from the gel under low power UV light for a time as short as 
possible. 

I elute the membrane with 1 mM Tris pH 8. 

I think I must agree with you that there's formation of single stranded 
DNA, since it cannot be a coincidence that all bands run at their 
respective half-sizes. Just didn't know that DNA strand separation could 
take place that easy.

Thanks for your help!

Maikel


"GysdeJongh" <jongh711 at planet.nl> wrote in
news:d4jmbo$6vd$1 at reader08.wxs.nl: 
> 
> Hi,
> so : before the QiaQuick purification the size on gel was OK (?)
> 
> Then you cut the fragments out of the gel . Did you use low power UV
> or even a Dark Reader ( this uses about 500nm and SybrGreen) to
> illuminate the gel for cutting ? If not , DNA is damaged very quick by
> UV !!!! 
> 
> Did you elute the Qia silica membrane with a buffer with sufficient
> salt ? Like TE or 10mM Tris ? If you elute the Qia column with WATER
> then your DNA will become single strands and will show about half the
> size on gel depending on the possible secundary structures of the
> ssDNA. 
> 
> If your DNA was not too large , < 1kb , then you might try to
> re-anneal the ssDNA back to dsDNA. If your DNA was much larger than my
> experience is that you end up with an insolulable "knot" of
> DNA-complexes if you try to re-anneal it. 
> 
> Hmmm your the second one with this problem today I'm aware of , the
> other was a college  :)
> 
> hth
> Gys
> 
> 




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