Christina - Those enrichment numbers seem a little high to me, so I would keep washing. As for my problem, it seemed to work itself out when I went from 5 copies/bead down to 2 copies/bead during amplicon sequencing (did not do a titration).
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Christina85... I am ABSOLUTELY sure it was the pipettor. I have photos and data to support this is a side-by-side experiment we did. We checked everything before hand as well... pcr plate, thermal cyclers, caps for the pcr plates, etc and we had Roche is 3 times. It wasn't until 454 came in with Roche and made this recommendation - now everything works like a dream. And there is correlations between our SVs and LVs (almost perfect). I can send you data / photos if you want to see them. Email me at [email protected].Comment
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One thing I've been doing to increase accuracy (I hope) is perform 2 volume measurements and 2 bead counts for each tube, and then use the averages. Measuring ~1200ul with a pipette is pretty tricky, and I've found that 2 back-to-back measurements, with the same pipette, tip, and user can give differences up to 50ul, and two back-to-back bead counts (using different Accuvettes and different 3ul aliquots) can give differences up to 500,000 beads. Little deviations like these can certainly add up and make troubleshooting difficult, so I'm doing it all in duplicate.Comment
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