High everybody
I am new to this community, it is great to have such a platform to ask questions!
My problem is the following:
I have performed a successful 454 Junior Run with a Rapid Library kit and Lib-L emPCR. The sample consists of PCR products and I calculated with 2 fragments/bead for the emPCR. It worked fine only that I had a quite many fragments which were concatenated.
Therefore I made another Run with 1.5 ul of adapters instead of 1 to increase the adapterNA ratio. I used a very similar sample and the same library prep kit but I got almost no enriched DNA beads. I repeated the emPCR but again only very few enriched beads. Then I redid the library and again the same problem. Afterwards I took 5 times the amount of library and I got more enriched beads but still not enough for sequencing. So I did another emPCR with 80 fragments/bead and I got just enough DNA beads. I got about 60'000 good reads which is much less than I got with the first run but the quality was good and the fragments fully sequenced.
As I had increased the AMPure bead to DNA ratio to not loose my shorter fragments, I thought the problem was that I had adapters in the library which lead to a too high measurement of concentration.
But now I made a new library with a size cutoff at 400 bp (seen on the Bioananalyzer) and again I had only about 10-20% of the enriched beads needed after emPCR.
The emulsions were not broken and the PCR machine, NaOH solution and all lab equipment was successfully used for another sequencing run in the meantime.
I wonder if the adapter ligation may not have worked properly so that only few fragments have both adapters. Is there any way I could check that?
Or do you have any other suggestions?
Thank you.
Joana
I am new to this community, it is great to have such a platform to ask questions!
My problem is the following:
I have performed a successful 454 Junior Run with a Rapid Library kit and Lib-L emPCR. The sample consists of PCR products and I calculated with 2 fragments/bead for the emPCR. It worked fine only that I had a quite many fragments which were concatenated.
Therefore I made another Run with 1.5 ul of adapters instead of 1 to increase the adapterNA ratio. I used a very similar sample and the same library prep kit but I got almost no enriched DNA beads. I repeated the emPCR but again only very few enriched beads. Then I redid the library and again the same problem. Afterwards I took 5 times the amount of library and I got more enriched beads but still not enough for sequencing. So I did another emPCR with 80 fragments/bead and I got just enough DNA beads. I got about 60'000 good reads which is much less than I got with the first run but the quality was good and the fragments fully sequenced.
As I had increased the AMPure bead to DNA ratio to not loose my shorter fragments, I thought the problem was that I had adapters in the library which lead to a too high measurement of concentration.
But now I made a new library with a size cutoff at 400 bp (seen on the Bioananalyzer) and again I had only about 10-20% of the enriched beads needed after emPCR.
The emulsions were not broken and the PCR machine, NaOH solution and all lab equipment was successfully used for another sequencing run in the meantime.
I wonder if the adapter ligation may not have worked properly so that only few fragments have both adapters. Is there any way I could check that?
Or do you have any other suggestions?
Thank you.
Joana
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