Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Qubit library quantitation variations

    Hi All,

    I am facing lot of variations in my library quantitation using Qubit dsDNA BR assay kit. Lambda DNA stock concentration which is provided by Sigma has a documented concentration of 400ng/ul and Qubit shows above 550ng/ul. What might be the root cause? Is it pipetting error or people tend to get variations from sample to sample?

    Please advise!

  • #2
    Hi, it could be pipetting errors, but more likely you simply have a bit of batch variation from Sigma so that it's not exactly 400 ng/uL. However, I don't have any experience with these stocks so anyone else can comment if they know.
    - Gina

    Comment


    • #3
      Hi,

      In our experience the Qubit BR assay is less accurate than the HS assay, so try doing a dilution of your DNA and using the HS kit if you have it. Our BR assay usually read higher than the HS, even if the stock DNA concentration fell within the range of both.

      One QC thing we've started is doing a spec of your Qubit standards, especially standard 2. We had one kit with problems and found it was almost 20% above the concentration written on the tube.

      Also mix your samples extremely well, this can make a huge difference especially if you're only measuring 1ul.


      -Liz

      Comment


      • #4
        Qubit (HS, BR)

        when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?

        Comment


        • #5
          When was the last time you ran the standards? If you're not running them every time then you're pretty susceptible to pipetting errors in your Q-reagent/buffer sln.

          Comment


          • #6
            I run the standard everytime and Qubit reads the right conc. for the standard but I got different readings for the same sample (20ng/Ml when HS kit was used) and (80ng/Ml when BR kit was used), have tried on other samples too and get different readings- it is not pipeeting errors so what is the explanation?

            Also, would DNA concentration decrease that much following freezing and thawing?

            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
            7 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, Yesterday, 06:07 PM
            0 responses
            7 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