Originally posted by bradfish
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I am having one heck of a time with old emPCR kits. I am having an enrichment nightmare where I simply cannot get any freaking enriched beads out of this (extremely low %, regardless of CPB). I hope these new kits can do better for me but you guys aren't exactly giving me confidence.
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Originally posted by bradfish View PostRight now I don't have time to post a lot of data, but I can tell you we are doing 1 cpb with new and old lots. Sequencing 400 bp amplicon, with 1/4 the amount of reccomended amp primer. We went from getting 400-600k enrichement consistenly for the last year to over 1 million on the last few runs with nothing changing except the lots.
As for the increasing enrichments with the newer kits, we haven't really noticed this, however our enrichments are higher on plates that sit for a while after the final emulsion shake. We find that these "false" higher enrichments tend to sequence poorly. Same CPB, Same reagents, just the 1st cup sitting out longer while the second is being aliquoted = poorer sequence. As a result, we have gone to processing one LV cup at a time, putting them on the thermalcycler ASAP once aliquoted. Processing the cups this way is much more consistent, albeit more time consuming.
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I've been loading one emPCR at a time as I figured letting it sit would be an issue. I usually pause when you add the amplicon to the oil, and just move forward with one from there all the way through the procedure, then add amplicon to the next oil and start from there again. Seems to work great.
Interesting thoughts on diversity of pool correlating with signal interference. What I am are pools of many different samples each containing at least a few different organisms, many containing much more. I am going to look into this correlation when I get time.
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If it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation.
Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments?
Our MID adaptor kit is over the expire date, could this explain the problem?
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Originally posted by boerm View PostIf it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation.
Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments?
Our MID adaptor kit is over the expire date, could this explain the problem?
YMMV on expired reagents. It just depends how precious your samples are. Other labs with unlimited budgets throw them out.
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Originally posted by DNA_Dan View PostThis might be a possibility, but the equilibrium favors dimer formation. If you want to test for this simply do an amplification on the final library and run it on a BA chip again. If those fragments are there you should pick them up.
YMMV on expired reagents. It just depends how precious your samples are. Other labs with unlimited budgets throw them out.
Dear DNA_DAN,
Thanks for your suggestions, do you have the sequence of the Lib-L primers to amplify this library?
Thanks,
Martin.
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See Application Brief # 001-2009 Entitled Unidirectional Sequencing of Amplicon Libraries Using the GS FLX TItanium emPCR Kits. (Lib-L) On the second page, Primer A and Primer B are for fusion primers. Just order these sequences up to (not including) the key pass sequence.
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