Issue with Titanium bead enrichment

[bbeitzel]

The last couple of times that I have enriched titanium sequencing beads after emPCR, I have had difficulty removing the sequencing beads from the enrichment beads. I bind the sequencing beads, wash with enhancing buffer until no more anti-enriched beads come off, and then recover with melt solution. What I have noticed is that after 2 (sometimes 3 or 4) treatments with melt solution, there is still a white layer attached to the enrichment beads. I am making up fresh 10N NaOH and melt solution.

Anyone else experienced this or have any ideas? I usually recover enough beads for sequencing, but I wonder how many I am losing, and why they are not coming off in the first place.

[LMcSeq]

I've heard that the white you're seeing isn't beads--it can be a precipitate from the NaOH that is added. Don't kill yourself to get this off, especially if you're collecting enough beads.
However, if there's a small amount of beads in the very bottom after collecting the melts, use a small volume of annealing buffer to collect the beads and add them right to the pooled melts.

[crndy52]

I have had the white layer you describe show up a couple of times in my lab. I found that it always happened when my bead recovery rate was way too high - sometimes as high as 30% or more. I have also noticed that when this happens some of the brown mag beads also come off during the washes.

[anujgupta]

These white beads are generally good beads, which you require. This is not NaOH precipitate. These are trapped beads, which you should remove from brown beads by gently pipetting up and down keeping the tube on MPC. Removed white beads should be tranferred to another 1.5 ml tube and again placed on MPC so that any brown bead present get separated. Summary: you should include those beads.

[Soulbee]

I found that the longer you leave the melt solution in the tube to get the sequencing beads off, the more they will bind back to the enrichment beads. I do my first melt treatment, transfer the sequencing beads to a new tube which i already have on MPC to do a second pelleting of any left over enrichment beads I may have gotten, then transfer from here to a new tube, spin and get rid of the melt. Repeat. pipetting up and down then waiting for the enrichment beads to pellet helps too. its okay to get some enrichment beads out with your sequencing beads out - that's what the second tube of pelleting is for. And after I add the annealing buffer, vortex, i place this back on to the MPC to get rid of any more enrichment beads if i feel that i still have quite a bit left over, then transfer the final sequencing beads and wash once more with annealing buffer.

[anujgupta]

They are not binding back. I mentioned.. they are trapped beads, you can separate them out by mild pipetting. Transferring to further tubes as you did also works. I give you one good idea. After melt, while washing by Annealing buffer, spin, but do not rotate and spin. Brown beads gets separated from while one which you can simple pipette out and discard.

[anujgupta]

They are not binding back. I mentioned.. they are trapped beads, you can separate them out by mild pipetting. Transferring to further tubes as you did also works. I give you one good idea. After melt, while washing by Annealing buffer, spin, but do not rotate and spin. Brown beads get separated from white ones which you can simple pipette out and discard.

[HMorrison]

see also http://seqanswers.com/forums/showthread.php?t=11978.