We are having problems getting our long-fragment shotgun library to amplify during emPCR. Titration using 3 different cpb ratios (12, 18, and 24) yields the same percentage enrichment (3-4%, which doesn't even give us enough beads for a 2-region PTP). A test aqueous phase PCR using Lib-L primers A and B also gives very poor amplification.
This is a library prepared by restriction enzyme digestion, genome reduction, gel purification and size selection, and finally adaptor ligation. This process has been reported to work in the literature, albeit with the FLX chemistry.
I'm really perplexed about the non-dependence of cpb on % enrichment. This might be a clue about what's going on. Anyone have any ideas about what is happening?
I haven't sequenced these beads. I had a look at the ssDNA left in the first melt supernatant on a RNA Nano Chip and saw lots of short fragments and only a smithen of 1000 base product.
Thanks.
Barry
This is a library prepared by restriction enzyme digestion, genome reduction, gel purification and size selection, and finally adaptor ligation. This process has been reported to work in the literature, albeit with the FLX chemistry.
I'm really perplexed about the non-dependence of cpb on % enrichment. This might be a clue about what's going on. Anyone have any ideas about what is happening?
I haven't sequenced these beads. I had a look at the ssDNA left in the first melt supernatant on a RNA Nano Chip and saw lots of short fragments and only a smithen of 1000 base product.
Thanks.
Barry
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