Butanol for breaking 454 emulsions?

[bbeitzel]

Anyone tried using 1-butanol for breaking 454 emulsions rather than isopropanol? I have been using butanol for breaking solution emPCRs, and it works very well for those. In my hands, isopropanol breaking in the standard 454 protocol is kind of a pain. The IPA does not seem to liberate the beads from the oil very well, so you have to do several IPA washes to remove residual oil. I am wondering if butanol would make the breaking step more efficient.

Also, the Ion Torrent protocol uses isobutanol, so I wonder if they are on to something.

[pmiguel]

I have not tried it. Would you use water saturated 1-butanol?

If you try it yourself, please let us know how it works. (Maybe try it for enrichment ePCRs so you are not risking as much?)

--
Phillip

[bbeitzel]

I have not been using water saturated isobutanol for breaking my solution emPCRs, and I get decent recovery. I might try saturated isobutanol on those to see if it increases the amount of product I can recover.

To test it for 454, I will probably just make an emulsion with leftover beads and oil and see what happens when I break with isobutanol. No sense going through the entire emPCR.

I'll post more after I've tried it.