Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Blue Pippen problems

    Is anyone else having problems with Blue Pippen's accuracy? Our error % from all our control DNAs tests range from 3-13%.

    One Example:
    Tight Setting(250bp)
    Bioanalyzer average size (bp) (282bp)
    12.8% error

    What is an acceptable error rate for the Blue Pippen?

    We have been using a 20bp offset for our samples (both amplicon and sheared gDNA) and BP control DNA. (So for a 390bp extraction, we use the 370bp tight setting.) This has been working for a while, but not anymore. We contacted Sage Science, who has been helping us a little, but we are still running through loops. Sage says our BP instrument log files are great and that it could be a cassette issue. They also think that since we are using PCR product, we may have single-stranded DNA that may be interfering with how we visualize peaks on the Bioanalzyer. This may be true but our sheared gDNA does not size accurately either.

    Sage Science suggests that, for every new sample type, we run a test run to determine the best setting. Does anyone do this?

    What other problems have you seen with the Blue Pippen? Is there a better alternative?

  • #2
    One has to take into account size distribution CV, reproducibility% and accuracy% for each Cassette type. Loading quantity, structure of DNA (blunt ended, Y shape, staggered ends) also can affect the size range distribution. On top of all of these also BA has its own specifications and deviation from them also will affect the sizing.

    Your example of tight setting at 370bp will collect fragments in 337-403bp. If you use range setting of 350-390 it will not collect 370bp as that setting is narrower than tightest setting possible for which the software gives a narrow warning.

    I agree with Sage explanation and their platform is much better than another one that I have tried.

    Comment


    • #3
      I know that to select small RNA libraries of ~150 bp and exclude adapter-dimer at ~130 bp you must use settings way different from expected (115 bp-170 bp collection range), so I am not surprised that a little optimization may be necessary.

      Regarding a better alternative, the only other thing I have tried is the E-gel, and Pippin is so much better it isn't even a comparison. I think the Caliper LabChip is probably a better comparison but I haven't had a chance to try it. I did find this Googling a little though.

      Comment


      • #4
        I had a similar result to the linked paper in kerplunk412 post comparing Pippin and LabChip.

        Comment


        • #5
          Thank you for responding

          Sage Science said that they like to see less than 10% error from the control DNA. This seems pretty high to me but if that's the case, then I guess we will have to test more than one setting for each sample type.

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Strategies for Sequencing Challenging Samples
            by seqadmin


            Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
            03-22-2024, 06:39 AM
          • seqadmin
            Techniques and Challenges in Conservation Genomics
            by seqadmin



            The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

            Avian Conservation
            Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
            03-08-2024, 10:41 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, Yesterday, 06:37 PM
          0 responses
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          49 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          66 views
          0 likes
          Last Post seqadmin  
          Working...
          X