Loss of resolution/retarded dye front on 3700

Phillip San Miguel pmiguel at purdue.edu
Fri Feb 18 16:28:26 EST 2000


    I've got a set of eight 96-well plates of Big Dye
terminator reactions that is producing a large number of
failures even though the signal strength is good. Most of
these failures (I'm getting 30 to 48 failures per run) are
correlated with low resolution chromatograms. These low-res
chromats also have retarded dye fronts. That is, when the
first peaks are coming off it looks like the rxns tanked
(produced no signal) but a few minutes later signal appears.
But the signal is blurred into long, somewhat noisy, hills
instead of tight peaks.
    2nd bit of data: this only happens on the right side of
the array. The left-most 20 capillaries are giving normal
data.
    I thought it was time for my array to be regenerated (92
runs since the last regeneration) but I got similar results
upon re-running these samples after the regeneration. I've
had pretty good runs on both sides of the regeneration with
other sets of samples (though mean read length as mesured by
Phred q>20 bases decreases about 50-100 bases across the
array (left to right) in a linear fashion.
    Probably something in these plasmid preps is interacting
with some other feature of my array or cuvette to produce
these results. Anyone got an idea what?
    I use Qiagen REAL preps plasmid templates--1/25 of the
entire volume. Up to this point it seemed unecessary to run
them on a gel or quantitate the yield. I do terminator
clean-up using BioRad P-30 ("Squeaky Clean") columns and
directly load the eluate onto my 3700 (after transferring to
clean PEBio 384-well plates.) Previous to this I have been
getting good results. (POP5 mean reads (phred q>20 bases) in
the 500's--sometimes topping out into the 600's. Often I was
getting less than 10% failures.)

Phillip San Miguel
Purdue Genomics Core Facility

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