Problems with 454 GS FLX Titanium medium volume sequencing
Hi,
I've been running my first ever pyrosequencing reactions recently, using all the kit and instructions provided by Roche for the GS FLX Titanium series. We're using 8 regions per plate, so the medium volume reactions, and we're using MID primers, with 12 primers per region (96 samples per plate). Our first plate, which we sequenced earlier this week, seemed to come out ok; however, the second plate, which I'm currently running, doesn't seem to have worked. The run is still progressing, but at the moment on the screen it is reading "0" for all control reads, and very low numbers (in the tens) for sample reads - so, seemingly, the machine is having problems picking up the actual samples themselves.
At the moment, I'm unsure whether to repeat the emulsion stage fo the protocol, or whether it's likely to be a problem earlier on, e.g. with the PCR (I don't think so - but it's possible). I was unsure about the emulsion stage, because I had a yellow information sheet saying that the lot of emulsion oil we used may produce a clear layer on top of the emulsion, so it was difficult to tell if the emulsions were broken. However, I had reasonable return of enriched beads; well over the 340,000 needed per region.
I'm a bit confused about why one run has worked and another hasn't. Can anyone give me any tips about which part of the process this is likely to be a problem with?
Thanks!
BL
I've been running my first ever pyrosequencing reactions recently, using all the kit and instructions provided by Roche for the GS FLX Titanium series. We're using 8 regions per plate, so the medium volume reactions, and we're using MID primers, with 12 primers per region (96 samples per plate). Our first plate, which we sequenced earlier this week, seemed to come out ok; however, the second plate, which I'm currently running, doesn't seem to have worked. The run is still progressing, but at the moment on the screen it is reading "0" for all control reads, and very low numbers (in the tens) for sample reads - so, seemingly, the machine is having problems picking up the actual samples themselves.
At the moment, I'm unsure whether to repeat the emulsion stage fo the protocol, or whether it's likely to be a problem earlier on, e.g. with the PCR (I don't think so - but it's possible). I was unsure about the emulsion stage, because I had a yellow information sheet saying that the lot of emulsion oil we used may produce a clear layer on top of the emulsion, so it was difficult to tell if the emulsions were broken. However, I had reasonable return of enriched beads; well over the 340,000 needed per region.
I'm a bit confused about why one run has worked and another hasn't. Can anyone give me any tips about which part of the process this is likely to be a problem with?
Thanks!
BL
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