emulsion PCR - (Jan/19/2016 )

Hi,

I'm trying to set up an emulsion PCR experiment but am having difficulty with the emulsion step. Does anyone have any experience with the classical stirring method?

a couple of questions on the emulsion:

1) how does the emulsion look before and after PCR? should they look identical?

currently my emulsions look milky before after the mixing step but after PCR, the micelles look as if they settled to the bottom of the tube. i.e. a cloudy oil phase on top, "white" phase at the bottom. is this normal? this settling effect also happens over time (~15 mins) if just left on the bench. I've also not have any success at amplifying my product as observed on gel. Non-emulsified PCR works fine.

2) does anyone have a working protocol for emulsion PCR with the Kapa2G robust hotstart taq kit?

No experience, but I would note that many of the PCR buffers and master mixes have detergents as a component. I would imagine that these would cause problems in an oil emulsion.