Cy5-labelled DNA migration

chovek69 via methods%40net.bio.net (by ivanoov from gmail.com)
Sun Jul 15 05:02:55 EST 2007


Hello all,

I am almost sure of the reason for the problem below but somehow I
wanted to hear an expert opinion. The story is that I used to perform
some MLVA (minisatellite) microbial typing with PCR Cy5-labelled
primers and denaturing separation on an ALFExpress II sequencer. It
was just fine. However now I am in another lab where I want to do the
same analysis by means of non-denaturing capillary electrophoresis
(CE) with UV/EtBr detection rather than laser.
It seems that the Cy5 introduces a serious mobility shifting besides
that most of the fragments appear as double bands. Of course I do not
apply a Cy5-labelled DNA ladder and e.g a fragment that is supposed to
be 480bp appear as 510bp. As I use a 5% DMSO in the PCR I wander that
it can be the reason for the the double banding. I will order non Cy5
primers but as I need some results asap is there some solutions for
the moment ? Maybe some protocol for degradation/separation of the Cy5
from the primer ?

Thanks in advance



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