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Old 01-18-2011, 06:33 AM   #1
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,313
Default Troubleshooting EZ Bead vs manual bead prep

We were having trouble getting a good set of beads with our new EZ Bead System. As it turned out, we probably were just not adding enough library.

We finally figured this out with the help of our FAS. The critical factor is that the EZ Bead, unlike manual bead prep, is not very linear on the low end with enrichment. With the manual method, if we thought we would get 1 billion beads, but we only ended up with 400 million then the beads we had would be of very high quality with high % numbers of on-axis (best+good) beads.

But if you get 400 million beads on an E80 EZ Bead run, then you probably want to throw them out! Apparently 200-500 million beads is about what you get from an E80 even with no good beads being produced.

We deposited 1 billion beads out of 1.6 billion enriched beads produced from an E80 run of our EZ Bead system. So far it looks good--the first ligation produced 81% best + good beads. Only 450 million of them, but 450-550 million looks like the de facto spec for a v4 slide. At least for the time being.

--
Phillip
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