I've been frustrated with emPCR for a while now because cpb ratios never seemed to correlate well between emPCR sizes. In my experience I found that a cpb ratio that yielded the desired 8-10% enrichment at the SV scale would give me anywhere from 25-50% at the MV scale. In addition, I seemed to get poorer data when using MV empCR kits. I could never figure it out until just recently.
I did a MV emPCR that looked somewhat broken when I took it out of the thermocycler. I've never had a broken emulsion before, so I was a bit surprised. I wasn't sure it was broken, either, because I had never seen one. There was just a thin layer of the aqueous phase at the bottom of the well with a few beads in it. To be sure it was broken I looked at a bunch of 1ul aliquots under a microscope. Sure enough, it was broken, so I threw it away. To make a long story short, I paid closer attention to the next couple of MV emPCRs, and that thin layer was there, but usually was only noticeable if you looked for it. Looking at aliquots from that bottom of the well versus the middle of the well showed that the emulsion was only partially broken; at the bottom of the well were vesicles with many beads and the rest of the well was filled with single-bead vesicles. I eventually figured out that the emulsion wasn't breaking, but instead was not being formed properly in the first place. Using the standard protocol there were a lot of vesicles in the emulsion with anywhere from 2 to several dozen beads. Icreasing the time didn't help, only increasing the speed of the last shaking step did. This last time I increased the shaking speed of the last shake to 20 Hz rather than 12 Hz and there were many fewer of the many-bead vesicles, and those that persisted were much smaller, but there were still some. The sequencing results were not great, either.
Has anyone else noticed inconsistent enrichment or sequencing results when doing emPCR at the MV size? If so, have you looked at the emulsion under a microscope and can you verify my experience? I'm trying to figure out if this is something unique to me, or if it is more wide-spread.
I did a MV emPCR that looked somewhat broken when I took it out of the thermocycler. I've never had a broken emulsion before, so I was a bit surprised. I wasn't sure it was broken, either, because I had never seen one. There was just a thin layer of the aqueous phase at the bottom of the well with a few beads in it. To be sure it was broken I looked at a bunch of 1ul aliquots under a microscope. Sure enough, it was broken, so I threw it away. To make a long story short, I paid closer attention to the next couple of MV emPCRs, and that thin layer was there, but usually was only noticeable if you looked for it. Looking at aliquots from that bottom of the well versus the middle of the well showed that the emulsion was only partially broken; at the bottom of the well were vesicles with many beads and the rest of the well was filled with single-bead vesicles. I eventually figured out that the emulsion wasn't breaking, but instead was not being formed properly in the first place. Using the standard protocol there were a lot of vesicles in the emulsion with anywhere from 2 to several dozen beads. Icreasing the time didn't help, only increasing the speed of the last shaking step did. This last time I increased the shaking speed of the last shake to 20 Hz rather than 12 Hz and there were many fewer of the many-bead vesicles, and those that persisted were much smaller, but there were still some. The sequencing results were not great, either.
Has anyone else noticed inconsistent enrichment or sequencing results when doing emPCR at the MV size? If so, have you looked at the emulsion under a microscope and can you verify my experience? I'm trying to figure out if this is something unique to me, or if it is more wide-spread.
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