Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • DNA concentration increased

    Hi,
    My sample concentration after PCR was 52ng/microlitre. After purification with beads it read 63,2 ng/microlitre. Is this normal? usually we lose some DNA after AMpure and this is the first time it happen to me. Can any one tell me what happened? should I repeat my Qubit measurement?

  • #2
    Are the before and after volumes the same?

    Comment


    • #3
      Maybe something's wrong with your measurement.
      You should try some other test methods (like Agilent 2100) for a validation.
      Last edited by woshiliangliang; 08-29-2013, 09:59 PM.

      Comment


      • #4
        Originally posted by dpryan View Post
        Are the before and after volumes the same?
        Yes, the before and after volume is the same

        Comment


        • #5
          Would the number of PCR cycle affects?

          Comment


          • #6
            Be aware that temperature of the Qubit reagents can alter results. So if you kept the tube in the Qubit housing or even in your hand longer or at all, it could cause a change. Also if you made up the reagents separately on those occasions they might be slightly different ratios.

            Another thought - was your PCR product centrifuged and mixed prior to your first reading? The DNA might not have been homogeneous within the sample, or even pipetting technique could be the answer.

            Comment


            • #7
              now I have measured the concentration of my samples after AMPure for two different genes and I got variable reading that I do not know how I can pool them.
              Gene A Gene B
              ng/μl Quantity in ng ng/μl Quantity in ng
              1 17.38 434 0.498 12.45
              2 8.92 223 0.904 22.6
              3 5.36 134 0.588 14.7
              4 13.8 345 0.578 14.45
              5 21.2 424 1.242 31.05

              Comment


              • #8
                Originally posted by dpryan View Post
                Are the before and after volumes the same?
                Hi, I have measured the concentration of my samples after AMPure for two different genes and I got variable reading that I do not know how I can pool them.
                Gene A Gene B
                ng/μl Quantity in ng ng/μl Quantity in ng
                1 17.38 434 0.498 12.45
                2 8.92 223 0.904 22.6
                3 5.36 134 0.588 14.7
                4 13.8 345 0.578 14.45
                5 21.2 424 1.242 31.05

                Comment


                • #9
                  The table isn't that easy to follow, but from what I can tell is you have lost DNA. Quite a lot in fact. 17ng/ul to 0.4ng/ul is significant. It might be a faulty batch of SPRI beads. We had a similar problem and a new batch solved the issue.

                  Comment


                  • #10
                    Sorry for the table it is a system problem. In fact the 17ng/ul is the conc of sample in gene A and the 0.4ng/ul is the con of sample in gene B.

                    I suspect the beads as well because my first experiment went much better. On the other hand the number of cycles in the PCR varied between the two genes. Could this be the reason? the gene with more cycles give higher concentration.

                    Comment


                    • #11
                      I thought it was an issue of concentration before and after a SPRI clean up?

                      If its a question about 2 separate PCR reactions it might be that you are not amplifying anything in your second reaction then.

                      What concentration of DNA are you putting into the PCR?

                      Comment


                      • #12
                        The DNa product are with a high concentration but I have diluted them to 8ng/µl, this is according to the protocol requirements. I used 25 cycles with PCR A but 20 cycles for PCR B. The 20 cycles worked very well with my previous experiment. I am afraid I might not be able to repeat these samples because I have a very limited primers material. That is why I wonder if there is a solution for this problem.
                        Last edited by MonaE; 09-05-2013, 08:16 AM.

                        Comment


                        • #13
                          What volume of 8ng/ul DNA have you used in the reaction?

                          I doubt this is an SPRI bead issue. It sounds like a problem with your PCR. Have you run a positive and negative control? It might be an issue with either your primers for gene B or the DNA you have put into the reaction.

                          Comment


                          • #14
                            I put 5 ul DNA in a total of 20 ul PCR reactions. Before PCR the concentration was between 5,4-8,54 ng/ul . After the beads they really varied.

                            Comment


                            • #15
                              So 40ng per reaction. I'd stop worrying about the beads. Its more likely the PCR. To be honest you're not getting great amplification from any reactions.

                              Take sample 5 in gene B for example if you put in 40ng after SPRI clean up you have 31ng. The PCR isn't working.

                              I'd check your PCR reagents. If your primers have worked well before just make a fresh batch, if not it might be worth designing new ones.

                              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
                              12 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, Yesterday, 06:07 PM
                              0 responses
                              10 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 03-22-2024, 10:03 AM
                              0 responses
                              52 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 03-21-2024, 07:32 AM
                              0 responses
                              68 views
                              0 likes
                              Last Post seqadmin  
                              Working...
                              X