missing 500 bp band w/ lambda hind3 (fwd)

Robert Preston rapr at MED.PITT.EDU
Thu Jul 7 08:53:26 EST 1994


> 
> Hi,
> We need some help.  We're running 1% and 2% gels in our lab using Lambda hin3 
> as a molecular weight marker.  We load 500 ng of lambda in our marker lane and 
> we don't get resolution of the 500 bp band when prestaining the gel with etbr.
> Do we need more DNA in the marker lane?  More etbr (we now use 2ul/50ml of 
> gel, this used to be enough).  Any comments/suggestions will be appreciated.
> 
Brian, have some help: the 500bp band contains 500ng x 0.5kb/40kb = 6ng of
DNA.  That is near the limit of detectability on agarose with EtBr staining,
and since the band is smushed-out wide because agarose has such crappy
resolution for fragments < 1kb, the signal gets lost in the background
fluorescence of the gel.  Use dual standard lanes: higher amounts of std
to detect the smallest frag (but then the big frags are overloaded) and
smaller amounts to resolve the big frags (but small frags invisible in that
lane).

Rob Preston
rapr at med.pitt.edu
> 




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