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[Automated-sequencing] DNA SEQ: 3730XL picket fence signal intensities.

Phillip San Miguel pmiguel at purdue.edu
Thu Nov 17 17:29:16 EST 2005


Mysterious signal intensity variation is plaguing us. Even capillaries 
show good signal strength, odd capillaries show poor signal strength. I 
also see this with ABI sequence standards, so it is not a problem with 
our reactions.

Here is a screen capture of a standard run:

http://data.genomics.purdue.edu/~pmiguel/technical/11-17-2005/image001.png


Not sure if it is relevant, but here is a screen capture of our spatial 
calibration:

http://data.genomics.purdue.edu/~pmiguel/technical/11-17-2005/image003.png


I hand-loaded the sequence standards myself into the A1 quad of an ABI 
384 well plate and heat sealed it myself. (By which I mean there are no 
robots involved.)

One of the service engineers attempting to troubleshoot this over the 
phone thinks it is a result of the array electrode being pressed against 
the bottom of the sample plate during electrokinetic injection. That, 
plus a slight difference in the length of the caps that maps even/odd. 
He had us try to back off a z-axis calibration in the 3730calib.ini file 
by 20 units. That resulted in the autosampler being unable to get the 
sample plate. So we tried 10 units. The autosampler worked okay then, 
but the "picket fence" phenomenon remained.

Although this is a new array, in looking back at old runs, I think I see 
the same phenomenon in previous arrays--just not as pronounced.

-- 
Phillip



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