>A spatial for each capillary will pass or fail based upon signal
>intensity. I don't think that at the level of passing or failing a spatial
>the alignment of the array should be an issue, although it is used to
>subsequently calibrate the bin for each capillary <can anyone confirm
>this?>.
Alignment of the array is part of the alignment issue. The spatial run floods
the capillaries with a single dye (red or blue) and the electrophoreses the
dyes out so they come out in a continuos stream. The streams are then used to
map the capilaries onto the CCD camera in the x-axis on the camera. The y-axis
is used for the spectral calibration. The pattern is important to help match
the capilaries correctly onto the camera so that the software will know which
lane is which during the following runs. The software expects that the
capilaries will fall into a certain range of pixels on the camera. If the
signals or pattern don't fall into the expected range the spatial will fail.
If the array is made incorrectly or is placed into the cuvette at an angle the
pattern will not match what the software expects and the spatial will fail. I
have seen an array that was crossed end for end (cap 1 at loading end was cap h
at detection end). You get a beautifull spatial with strong peaks that fails
because the patern is reversed.
Alignment of the laser is also an important factor. you can get a weak signal
that may cause an excessive number of caps to fail, or you can get the smile or
left to right fading issue.
Hope this helped.
Steve
Director DNA Sequencing Core @ Caltech
former ABI service engineer
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