So the basis of ePCR is that every DNA template will ideally be isolated with polymerase in a water droplet, in an oil phase, creating a “PCR microreactor.”
How will this method affect synthesis throughput?
Did the preliminary tests of ePCR with the current synthesis/sequencing pipeline show any potential pros or cons?
Besides ePCR, what other ways is the Das lab looking into further development of the synthesis and sequencing pipeline?
1. How will this method affect synthesis throughput?
emulsion PCR delays library amplification – the first step of our experimental procedure – by one day, and can occasionally give worse yields.
2. Did the preliminary tests of ePCR with the current synthesis/sequencing pipeline show any potential pros or cons?
All of our ePCR tests have shown distributions of sequence lengths in better accord with designed dsitrbutions. Solution PCR typically upweights small fragments.
Important note: We used ePCR to amplify some of our initial libraries in 2013, but then switched to solution PCR for speed later in the year. I will ask Ann to annotate this on our google doc of experimental rounds.
3. Besides ePCR, what other ways is the Das lab looking into further development of the synthesis and sequencing pipeline?
We will probably not spend much more time on the development of this pipeline. We would rather focus our attention on new readouts with higher throughputs and the prospect of testing functional RNA devices (multi-input switches).
There is, however, a chance that we will retake some experimental rounds with poor signal-to-noise at some point next year – this will depend on whether we (or players) develop algorithms that will benefit greatly from having all the data with high signal-to-noise.
4. If the amplification is isolated in separate “bubbles”, does this affect transcription, chemical modification and/or reverse transcription as well? I’m specially interested in knowing whether dimerization experiments are still conceivable or not.
The amplification is in separate bubbles, but we then destroy the emulsion,and extract the ‘aqueous phase’ (inside the bubbles) to purify. Subsequent steps are not conducted in emulsion.
@nando and @brourd. Is there a page on the Wiki that goes through the entire experimental protocol? If so, we might want to update it with an explanation of emPCR. If not, we probably should have such a page, and we can ask jnicol to link to it from the lab puzzle page.
Thank you for the reply, Rhiju!