6 Replies Latest reply on Feb 19, 2019 6:32 PM by linche01

    Mx3000P additional peak

    linche01

      Hi,

      My customer increased the primer concentration and got an additional peak (marked with the red square below) near the main peak. Would you kindly let me know what is the possible reason? Please see the image below. Thank you so much!

      Mx3000P peak.png

        • Re: Mx3000P additional peak
          agrafsky

          Hello,

           

          While we do have a general idea of what might be causing this additional peak, we would be better able to support your customer if you could send the original data file (.mxp) in which these results were observed to qpcr@agilent.com<mailto:qpcr@agilent.com>.

           

          Could you please forward this file?

           

          Thank you,

           

          Albert Grafsky

           

          Manager,

           

          Technical Services

           

          Agilent Technologies, Inc.

           

          T: +1 800 227 9770    |    M: +1 858 229 1118    |     www.agilent.com<http://www.agilent.com/>

           

          F: +1 858 597 0619    |    al.grafsky@agilent.com

           

           

           

           

           

           

          Follow Agilent on:

           

               

            • Re: Mx3000P additional peak
              agrafsky

              Before seeing the original .mxp file, one comment that can be made is that higher temperature dissociation peaks such as the one seen here have frequently been associated with the presence of rare splice variants of the target sequence. Alternatively non-specific product formation, due to the primers hybridizing to sequence other than the intended target, has also been known to cause such peaks. Since you indicated that this peak appeared after the customer increased their primer concentration, it seems that the second cause is the more likely.

              • Re: Mx3000P additional peak
                linche01

                Thank you for all your comments and explanations, Albert! I'm asking for the .mxp file.

                • Re: Mx3000P additional peak
                  linche01

                  Thank you, Albert. I'll send the file right now.

                  • Re: Mx3000P additional peak
                    linche01

                    Hi qPCR support team,

                     

                    I submitted a question on the community site, about an additional peak from Mx3000P. Following dear Albert’s suggestion, I send .mxp files.

                     

                    The additional peaks appears in the file Jan 30 evening, 2019, well C1, D1, G1, F1.

                     

                    The customer provided additional information. He is using a kit from a local vendor, and the qPCR reaction is set up as follows,

                     

                     

                      1.  The 2 experiments, Jan 30 and Jan 30 evening, used same qPCR set up, difference is the anneal and extend time at 60℃. Jan 30 used 15s for annealing/extending, and Jan 30 evening used 30s.

                      2.  Template. For both experiments (Jan 30 and Jan 30 evening), well A1, B1, E1, F1 used undiluted cDNA, while C1, D1, G1, H1 used 10X diluted cDNA as template.

                      3.  Primer concentration. A1, B1, C1, D1 used 1ul primer, while E1, F1, G1, H1 used 1.25ul primer.

                     

                    Sample Well

                     

                    cDNA concentration

                     

                    Primer volume

                     

                    Anneal / extend time

                     

                    Result

                     

                    A1, B1 (Jan 30)

                     

                    Undiluted

                     

                    1ul

                     

                    15s

                     

                     

                     

                    C1, D1 (Jan 30)

                     

                    10x diluted

                     

                    1ul

                     

                    15s

                     

                     

                     

                    E1, F1 (Jan 30)

                     

                    Undiluted

                     

                    1.25ul

                     

                    15s

                     

                     

                     

                    G1, H1 (Jan 30)

                     

                    10x diluted

                     

                    1.25ul

                     

                    15s

                     

                     

                     

                    A1, B1 (Jan 30 evening)

                     

                    Undiluted

                     

                    1ul

                     

                    30s

                     

                     

                     

                    C1, D1 (Jan 30 evening)

                     

                    10x diluted

                     

                    1ul

                     

                    30s

                     

                    Additional peak

                     

                    E1, F1 (Jan 30 evening)

                     

                    Undiluted

                     

                    1.25ul

                     

                    30s

                     

                     

                     

                    G1, H1 (Jan 30 evening)

                     

                    10x diluted

                     

                    1.25ul

                     

                    30s

                     

                    Additional peak

                     

                     

                    I agree that non-specific production formation possibilities increased due to template dilution, primer increasing, and annealing time increasing. Also increased extending time enables production of longer fragments. Correct?

                     

                    Thank you!

                     

                    Best regards,

                    Lin

                    • Re: Mx3000P additional peak
                      linche01

                      Dear Albert,

                       

                      Fully got it, thanks so much for your support.

                       

                      Best Regards,

                      Lin

                       

                      Sent from my iPhone