[pmiguel]

I am pretty sure there is a considerable amount of PCR product in the aqueous phase (not attached to the beads). I think some unanchored B primer is included in the PCR primer cocktail. Also, if all the amplicons end up attached to the beads, I don't see the reason to be so vigilant about amplicon contamination of the lab during emulsion breaking.

I agree the isopropanol used for breaking would be an issue though.

How about taking an aliquot of the beads (prior to melting with NaOH), heat denature to release the second strands, pull down the beads with a magnet and pull off the second strands and run them on an RNA chip? That way no need to do the ethanol precipitation.

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Phillip

[ajthomas]

You may be right about there being some B primer in the amplification primer mix. If so, I wasn't aware of it. Either way, there will be some PCR product in the aqueous phase (single stranded if no unanchored B primer, double stranded if there is unanchored B primer). Beside the aqueous phase, the supernatant from the melt step is a also a contamination risk. All of those second strands that are melted off the beads (and whatever PCR product is in the aqueous phase) are at a high concentration and a fantastic template for emPCR.

As for your suggestion of heat denaturing the second strands, I suppose you could do that. You would have to get them pretty hot (94C) to melt them off, but it should work. I'm not sure what you would gain by doing so, but it shouldn't hurt.

[pmiguel]

QC step. If run these 2nd strand on a RNA Pico chip and see lots of low molecular weight stuff you would suspect adapter dimers in your library. You also might get a general idea of how heavily templated the beads are over all.

I am just looking for ways to shine a light into this darkened room of the protocol.
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Phillip