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> Sequencers: I was wondering if anyone out there has seen this problem
> (and if
> you have, what causes it and how did you solve it):
>
> Sequencing M13 shotgun clones on 377s (we don't see the problem on
373s)
> and
> the sequence goes along well for 100 to 150 bases or so and then
fades
> (very
> low peak height) only to return with a good peak height about 100
bases
> farther down. If our signal intensity is >400, this doesn't cause a
> problem,
> but if the base signal intensity is <<200 we lose some sequence
data.
>
> This was a severe problem when I took over the sequencing operation a
> couple
> of months ago, but new plates and long ranger gels seemed to solve
it.
> However, this morning, there were about 4 lanes that showed this
pattern
> (the
> rest of the gel was fine, as a matter of fact we had alignable
sequence
> out to
> 940 bases on a couple of lanes (most had quality bases out past 750).
> Really!)
>
> So, if anyone has any experience with peaks fading out and then
coming
> back, I
> would appreciate hearing some words of widsom on the subject.
>
> TIA
>
> SRLasky
Hi Stephen.
We've had the same problem for a number of months before finding a
solution. Apparently the signal fading is caused by dried acrylamide on
the plates which accumulates between runs. We used to only rinse the
plates of our 377 after the run and would thoroughly wash them just
before using them again.
Apparently thats not the way to go. We now wash them with alconox as
soon as possible (wich usualy as soon as convenient) and rewash them
completly before use. Sounds tedious but its better them having to
rerun samples that are missing some data because of signal fading.
To get rid of the signal fading you already have on your plates, just
soak them overnight in 2M NaOH and then 20 minutes in 2N HCl to
neutralise them. Then rinse them thoroughly in water and wash them well
with alconox.
We have treated are plates like this 6 weeks ago and have not seen the
sinal fading problem reapear.
Hoping it will solve your problem too.
Sylvie Cornibert
Molecular Genetics lab,Algene Biotechnologies, Montreal, Canada.
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