High background on A peaks following G peaks

Anthony Anthony
Wed Nov 20 11:33:50 EST 1996


In article <56t421$ehh at net.bio.net> Steve Mullen,
mullen at lenti.med.umn.edu writes:
>Lately I've been experiencing very high background peaks under A's when
>preceeded by G's. It has been happening in the control as well as the
>client reactions. I am using the ABI Dye terminator cycle sequencing ready
>reaction kit for all of my work. Has anyone been experiencing this and
>found a correction?



The small peaks under a high A peak are most likely "pull-up" peaks.  The
gel
 matrix that the engineer installs at setup of a new instrument corrects
for
 the spectral overlap between the different dyes (giving a clean single
colour
 peak for each dye), but over a certain limit the matrix cannot cope (the 
 camera is outside its linear range) and the spectral overlap causes
bleedthrough, 
 meaning that a red signal will be interpreted as a red plus a small
amount of green 
 peak, for example.  This gives pull-up peaks.  The reason you see them
especially under
 As following Gs is that As folowing Gs are particularly strong, and thus
most likely
 to exceed the instrument's linear detection range.  If you see excessive
pull-up, it
 could be that your gel matrix is incorrect.  You can make your own with
ABI's matrix
 standards, but I'd phone ABI if I were you.
 
 Hope this helps
 
 Anthony.



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