Dear Friends,
Our 373XL 24 cm wtr is giving us poor data. This has been a gradual
problem which has been getting worse. It manifests itself as noisy
background. We can look at our older controls and see no background
peaking on the EPT out to 800 bases with PMT gain at 1100. Lately, the
background peaking is so high that background can become as high as the
real base peak, giving rise to an "N" or a miscall at only 300 bases with
PMT gain at 1000.
On the gel, the problem can be visualized as bands that are spread out so
that one base is on top of the next. At the extremes (600 to 800 bases),
you can see two or three different colors on the expanded view by turning
off all but one color on the control panel. The individual bands start to
become a lot fuzzier than normal the further up the gel you look.
ABI has come in four times to try and fix the problem. A laser was burned
out and they thought that this plus some misalignment was the cause. The
background noise was definitely worse on the right side. Samples run on
the left side seemed to look better. After the fourth fix, the bias was
reversed. Samples on the right side definitely looked better. I ran the
same pGEM control in lanes 1 and 64. Lane 1 had 14 mismatches in 600 bases
while lane 64 had 4 mismatches (unedited). We had been averaging accuracy
of 99% in 800 bases (8 mismatches on the average unedited).
>From what I have seen, I believe that this is an optical problem. My guess
is that it is some type of reflection or refraction due to laser alignment
or depth setting. This is not some primer or template problem since we
have run the same finished sequence taken up in loading dye on another
sequencer and it gave excellent results. We have used new TBE, different
acrylamide and new APS. Any help would be appreciated.
Thanks
Werner
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