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  • Aniki
    replied
    Originally posted by exon View Post
    Anyone also work with high cpb with improved enrichment percentage?

    We also suffered from low enrichment problem, mostly below 2%. The template that we are using is amplicon with fusion primer (with adaptor and barcode). We have checked the template with bioanalyzer and KAPA qPCR first. Then we used 1.7 cpb in the LV Lib-L emPCR. The enrichment is lower than 2% that we suspected the cpb not optimize, emPCR failed or the problem of the template? Then we went for SV using various cpb from 0.25 to 17, again the enrichment was <2% while the positive control (from previous work fine library) is OK but cpb increased from 1.7 to 14.6. We have already repeated SV in three times and used up the reagents.
    Unfortunately I cannot help you as I still do not know what causes this. I can only tell you that for me personally this is a trial and error situation still (since months now). Good luck.

    Leave a comment:


  • exon
    replied
    Anyone also work with high cpb with improved enrichment percentage?

    We also suffered from low enrichment problem, mostly below 2%. The template that we are using is amplicon with fusion primer (with adaptor and barcode). We have checked the template with bioanalyzer and KAPA qPCR first. Then we used 1.7 cpb in the LV Lib-L emPCR. The enrichment is lower than 2% that we suspected the cpb not optimize, emPCR failed or the problem of the template? Then we went for SV using various cpb from 0.25 to 17, again the enrichment was <2% while the positive control (from previous work fine library) is OK but cpb increased from 1.7 to 14.6. We have already repeated SV in three times and used up the reagents.

    Leave a comment:


  • Aniki
    replied
    I am having horrendous 1-3% enrichment values for anything cpb I use. Maybe I should try really high...

    Leave a comment:


  • Anthony.287
    replied
    Nope. That particular library has been titrated from 0.05 all the way to 32 cpb, doubling the DNA amount at each step. 16 cpb had ~5%, and 32 cpb 7.7%.

    Leave a comment:


  • Aniki
    replied
    32CPB?????? WHAT?

    are you kidding me?

    Leave a comment:


  • Anthony.287
    replied
    Lib-L Reagents - 93848220, 93867020, 93869820
    Lib-A Reagents - 93867520, 93856620
    Oil - 93840120, 93852520
    Recovery Reagents - 93871620, 93874520, 93871620

    I've had low enrichment percentages with all of these lot numbers. The most recent titration I've done (an amplicon pool, concentration determined with qPCR) yielded 7.7% enrichment at 32 cpb, which seems insanely high. Now that I've found a working cpb, I've received the new Lib-A reagents.
    Any suggestions?

    Leave a comment:


  • Aniki
    replied
    I believe I have had at least 2 or 3 different lot numbers that showed low enrichment numbers or low raw well numbers.

    The most recent lot numbers I have got are these:
    emPCR Kit 93856620
    Bead Recovery Kit 93871620
    Packing Beads 93866120
    Supplement CB 93851920
    Pre-wash Tubes 93866220
    Titanium Buffer & Pre-wash Buffer 98302056
    PTP Plate 93871720
    Sipper Tubes 93874720
    Sequencing Kit 83114
    Buffer CB 93874020

    Leave a comment:


  • LAH
    replied
    These are the lots of reagents that I was using during our erratic enrichment problems (still haven't received replacements):

    emPCR Kit Reagents Lot 93840420 Exp. 06/2012
    emPCR Lib Emulsion Oil Lot 93831020 Exp. 01/2013
    emPCR Kit Bead Recovery Reagents Lot 93837120 Exp. 01/2013

    THESE lot numbers are what I used for successful enrichment:

    emPCR Kit Reagents Lot 93845620 Exp. 07/2012
    emPCR Lib Emulsion Oil Lot 93836520 Exp. 03/2013
    emPCR Kit Bead Recovery Reagents Lot 93866020 Exp. 04/2013

    Good luck!

    Leave a comment:


  • GraemeFox
    replied
    Would somebody please give an example LOT number that is giving these very low enrichments?
    I am working my way through my stock of emPCR kits and I would like a little bit of warning if I am suddenly going to get to this problem myself.
    Thanks

    Leave a comment:


  • genomeseeker
    replied
    2 weeks after my last comment and I'm now experiencing the low enrichment issues (1-3%) on the heels of incredibly high enrichment/inconsistent enrichment percentages. I guess I'm getting into the same lot numbers that everyone else has been using...

    Leave a comment:


  • Aniki
    replied
    Unfortunately I can only add to the displeasure here. Since a few months now, we are battling one of several things that happen in our full scale LV emPCRs.

    1) low raw well numbers (very low)
    2) short read lengths, even though the library has an average bp value of 750+ and very clean no primers.
    3) very very low enrichment values despite having good titration values (2-3% enrichment)

    The low raw well and low enrichment values have happened so far only to our Lib-A runs. The low read length has been happening to both Lib-A and Lib-L.

    I don't know what to do, no one really knows whats the problem. I am a little scared to do anymore runs.

    Leave a comment:


  • genomeseeker
    replied
    I've been battling the opposite issue for the past couple of months. We're getting incredibly high recoveries and very inconsistent recoveries. When this started we were told by tech support there were some possible suspect recovery reagents out there that could be contributing to the issue. We purged suspect lot numbers, got replacement reagents and have settled with using much lower cpb; for our fusion amp libraries we've had to reduce 10-fold. For our rapid libraries we've cut the cpb by half (at least) to get good recoveries.

    Leave a comment:


  • Anthony.287
    replied
    I've had the same problem with rapid libraries and amplicon pools, both Lib-L and Lib-A. I use the Kapa kits for the final quantification for determining concentration going into emPCR. It reports a lower concentration than the FAM adapters or pico green, which is to be expected.
    What really baffles me is how well things were going here just 3 months ago, and the only thing that has changed here is the libraries I'm working with. Some of these were made with the same primers that previously worked quite well, and now they aren't working at all.
    Our FAS has been great about support and sending replacement kits; she even sent a rapid control library, which worked exactly as expected. Oh well, I'll give it another shot next week and report back.
    Have a great weekend!

    Leave a comment:


  • LAH
    replied
    Hi Anthony,

    Yes, we had a similar experience last month with several rapid libraries that looked great. But when we started to do titrations, we would get almost no enrichment. We also kept bumping up our cpb ratios and we would get erratic results: no enrichment on lower and upper values and extreme over-enrichment with the middle value - made me crazy. Finally I just broke out new lots of oil, emPCR reagents and bead wash buffers and everthing worked as it should. Our account manager told us to just send in the lot numbers and ask our tech support for replacement reagents - we are still waiting for those.
    This has become our new M.O. - if something doesn't work after a couple of attempts - switch to new lots of reagents and ask for replacements.
    Good luck!

    Leave a comment:


  • RCJK
    replied
    What types of libraries have you been titrating? Also, how to you quantify the input? With the FAM on the adaptors or with Kapa qPCR?

    Leave a comment:

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