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  • Bead Enrichment - infinite washes

    Hey,

    has anyone had issues with the enrichment step where the white beads literally ALL attract and stick to the brown beads. They congregate in a way where they are not in between the brown beads but instead coat the brown beads from the outside. Their attraction is relatively weak and the white beads break off very easily, however, this leads to quite a few washing steps (40+).

    One thing of note is that the library I received was not very clean with both genomic and adapter contamination quite noticeably in a bioanalyzer.


    If anyone has had this problem and a solution please give help!

    Thank you so much.
    Last edited by Aniki; 06-10-2011, 10:51 AM.

  • #2
    I had it once, I figured out that I had enrichment rate of 80% WOW. I think you need to check of primer dimer in case if it was amplicons. in general it must be copy number issue. do another emulsion with 70% less CPB, if you used 2 then use 0.5 cpb.

    Mo

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    • #3
      Hi Aniki

      White beads that actually covers the brown beads are no worth. They need to be washed off anyway. If you invert the MPC couple of times very slowly they will get dissolved in the supernatant and from where they can be removed very easily.

      thanks

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      • #4
        Aniki,
        We've been seeing this problem a lot lately and the only solution seems to be to repeat the initial melt step. Will try to get more details. But the first time around, we would have calculated an 80-100% enrichment; it drops to 8-20% range (normal) on the second round. Seems like a lot of steps are not working the way they used to 2 yrs ago. We use LibA kits primarily, but have also experienced it with LibL.

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        • #5
          We had same problem with Bead Recovery Reagent LOT93859020. You need to call tech support and request replacement.

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          • #6
            Bead enrichment problem--picture

            See total binding on first try.

            Problem lots (bead recovery kit lot #):
            93866020
            93871620
            93859020
            93837120
            Attached Files

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            • #7
              Yesterday we had a meeting with our FAS and he said lot 93837120 was fine and we should keep using it, and no longer use 93848420 and 93852420. All the boxes we have in our lab belong to the lots I mentioned. Looks like there is a problem with much more lots than we originally thought.
              Last edited by MissDNA; 04-13-2012, 02:27 AM.

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              • #8
                I experienced this yesterday and today; Lot number 93859020. Yesterday I was working with 3 samples each amplified (1xSV) using 1 and 3 cpb with Lib-A A. Today I was working with the same 3 samples amplified (2xSV) using 2cpb with Lib-A A. Final enrichment for *all* of the titration samples was around 10%. Today I called Roche, and didn't finish enriching. They will be replacing the kit. Unfortunately my remaining breaking kits have lot number 93866020, which was not one recognized by the person I spoke with at Roche as having problems, but is listed in this thread. I will setup one sample at 1 and 3 cpb as I had the first time and see what happens.

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                • #9
                  Originally posted by erikt View Post
                  I experienced this yesterday and today; Lot number 93859020. Yesterday I was working with 3 samples each amplified (1xSV) using 1 and 3 cpb with Lib-A A. Today I was working with the same 3 samples amplified (2xSV) using 2cpb with Lib-A A. Final enrichment for *all* of the titration samples was around 10%. Today I called Roche, and didn't finish enriching. They will be replacing the kit. Unfortunately my remaining breaking kits have lot number 93866020, which was not one recognized by the person I spoke with at Roche as having problems, but is listed in this thread. I will setup one sample at 1 and 3 cpb as I had the first time and see what happens.
                  I will be very interested to know the results. Thanks.

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                  • #10
                    Originally posted by erikt View Post
                    Unfortunately my remaining breaking kits have lot number 93866020, which was not one recognized by the person I spoke with at Roche as having problems, but is listed in this thread.
                    I too will be interested in your results. Everything I had was either 93848420 or 93859020, so I called Roche and they replaced them all, but what they sent was 93866020. I've used that lot a couple of times now. It seems to be similar to 93859020, although to a lesser degree. I have to do a lot of washes, but by the end the beads seem okay. I plan to do the sequencing run on those beads tomorrow; I hope they work okay.

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                    • #11
                      I'll post the results.

                      AJ, if you do sequence those beads I'd definitely be curious to know the results.

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                      • #12
                        Roche is going to replace all our 93848420 and 93852420 boxes. They said we should discard all the 8420 boxes but we should save the boxes from 2420 that present normal quantity of beads to be used in case of need.

                        What we have been getting so far is 93866020, which I hope offers no problems.

                        We still have a few boxes of 93837120, which is also listed here but Roche says is fine.

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                        • #13
                          Unfortunately my results today were somewhat inconclusive. 1 cpb yielded 2.5%, and 3 cpb 2.9%. However, the first wash did remove the largest portion of the beads, and far fewer washes were necessary, so that is encouraging. I received my replacement kits today; those are also lot 93866020, so I have little choice but to carry on. I'll follow up if I run into any trouble with this lot.

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                          • #14
                            We did a LV recovery today using lot 93866020 for the first time. It was necessary 12 washes while normally we do around 6-7. At the end we got around 2.5 million beads per cup, which is perfect for sequencing. Sequencing will happen tomorrow. We will be using XLR70. I am looking forward the results and certainly will check the filters.

                            Still waiting on our FAS response to see if this lot has a flag or not.

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                            • #15
                              FAS told me the lot was not flagged. We did the sequencing run and what was noticiable was a very low raw well number, which made me remember a post I saw on this forum:

                              "93852420 we had problems with low % recovery (below 5%). If we used a different kit with the same cpb, the emulsion would work every time. I was informed that the problem with this lot number though does not cause emPCR failure but, and I quote, "Rather than observing emPCR failure, this particular lot number was associated with a low raw well number in analysed seq runs"."

                              Once again I will contact support! This has become a daily routine for me. So many problems!

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