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Old 07-20-2011, 07:30 AM   #1
suefo
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Location: Ann Arbor, MI

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Default Poor distribution of "passed" wells on GS Junior

Has anyone else looked at the well images for their Junior runs? We have been getting really low % Passed Filter (like 10%) on the Junior. When we went back and looked at the actual images and the individual identifiers (no key, library, control and passed wells) we noticed that all of our passed wells were in the top third of the PTP for those containing DNA (Amplicons). Every run looked like this - accept for those with control beads only. When running the control beads only, we got even distribtion across the entire region of the PTP.

Just wondering if others have seen this. I think I uploaded a bmp to show what this.
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Old 07-20-2011, 07:38 AM   #2
suefo
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Default Perhaps a jpg will post better?

Trying to get image up. I apologize.
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File Type: jpg dna vs control bead distribution.jpg (73.5 KB, 42 views)
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Old 08-05-2011, 02:35 AM   #3
Palecomic
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Default

I've seen this once on our Junior, and it was also about the top third that worked and the bottom third that failed. This turned out to be due to the centrifuge we were using, the Eppendorf 5450 (I think that's the number!) smaller centrifuge for the BDD doesn't hit vertical and beads physically leaked out the sides during spins.

There was an obvious bead residue left inside the centrifuge, so I don't know whether it's the same cause in your case - they're quite noticeable brown smudges!

The solution Roche gave us was to stick tape over the ports on the BDD whilst using this centrifuge (apparently they no longer recommend this model). We use our Beckman instead which doesn't have this issue.
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Old 08-05-2011, 04:45 AM   #4
suefo
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Palecomic - thanks for the reply. Yes... I believe it was our centrifuge. We just completed 2 more JUNIOR runs where we paid special attention to the position of the PTP in the centrifuge. We made sure the loading end (the larger orifice) was always to the outside, so during rotation there wasn't loss of material. We are now getting great distribution and many more passed wells. We have an Allegro 6R from Beckman.
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