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-   -   Bubbles and XD slides (http://seqanswers.com/forums/showthread.php?t=7317)

pmiguel 10-13-2010 05:54 AM

Bubbles and XD slides
 
We recently had a case were our long mate pair library beads were looking great for the first tag (R3) but terrible for the second (F3). We were unable to finish the F3 run. In looking at the slide we found that most of our beads had been stripped off the slides in both flow cells.

The new, XD, deposition method/chemistry had been up to this point something of a disappointment for us. We were unable to get much past 500 million beads deposited on a slide -- far short of the 700 million that is specified in all marketing for the v4 instrument. Also, it seemed subject to strange artifacts near the injection ports -- circular areas that seemed scoured and were depleted in bead numbers.

But these issues paled in comparison to the problem described above. We were perplexed as to the source of our new issue. At first no answer seemed forthcoming. But then we began hearing that XD depositions are particularly susceptible to bubbles during a run. I am not clear on the nature of this susceptibility -- whether the bubbles just mechanically knock the beads off, or if they alter the bead linkage chemistry, leading to beads being washed off.

Either way, as long as the fluidics of the SOLiD remain more-or-less bubble free -- this should not be a problem. After this disastrous run failure, our service engineer ran a fluidics check for us and found an issue in an unexpected place. He visually confirmed that the lines were not pumping bubbles into the flow cells. Yet after fluidics that involved adding liquid from the storage buffer bottle, the flow cells consistently filled with bubbles.

This behavior went away when the cap for the storage buffer bottle (labeled "8b" in Appendix E of the System Instrumnet Operation Guide) was replaced. Our guess is that the fitting the connects the fluid tubing outside the storage buffer bottle to the straw that dips down into the storage buffer inside this bottle had somehow become clogged, and was therefore pulling a vacuum on the flow cells under some circumstances, causing air to be drawn in through the gaskets. This fit our observation that the first tag looked great, but the second tag was terrible because prior to the pause between tags, the instrument pumps storage buffer into the flow cells.

One could also imagine scenarios where this might crack slides were the gaskets to hold firm. For example, in cases where the ambient temperatures were a little higher, the gaskets might seal more effectively because they were less rigid. Then the sustained pressure differential across the glass slide might favor formation of slide cracks.

Something to keep in mind...

--
Phillip


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