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  • QIAquick Absorption Peak at 230nm rather than 260

    Hello,

    Each time, I used QIAquick gel extraction kit to extract the size selection product, the OD 260/230 is extremely weird (very low, close to 0), and 260/280 is a little bit higher (2.0-2.1). I found the absorption peak was moved from 260nm to 230nm, but the DNA should be extracted. I guess the remaining salt (such as EDTA) causes this, but I've tried to optimize the gel extraction step for several times (such as adding 6X volumes of QG, extending incubating time for QG, standing 5min after adding PE, ...), but they didn't work.

    The colleagues at lab also have this problem, but it didn't affect their TOPO cloning or Sanger sequencing.

    I have no idea if such weird ratio will affect some steps in the Illumina paired-end library preparation, such as Phusion PCR reaction or Cluster generation?

    Anyone has such experience?

    Many thanks,
    SK

  • #2
    It's the guanidium-SCN in the QG. An extra (or even a third) PE wash can help. In my experience it can affect efficiencies of sensitive enzymatic reactions, but is not a huge problem for amplification based preps.

    Comment


    • #3
      If it is a peak at 230 nm, that probably is a guanidium salt. But if it is somewhat shorter it may be acetate (eg, sodium acetate). At high concentrations absorbs there. (I can dig up the reference and post it here, if anyone want me to...)

      The difference is probably crucial. Sodium acetate is just a salt, but guanidium salts are powerful denaturants.

      --
      Phillip

      Comment


      • #4
        I' sorry, it's the peak shorter, it's just significantly higher at 230 than 260. (please see attached)

        So the sodium acetate should not be a problem, am I right?

        Originally posted by pmiguel View Post
        If it is a peak at 230 nm, that probably is a guanidium salt. But if it is somewhat shorter it may be acetate (eg, sodium acetate). At high concentrations absorbs there. (I can dig up the reference and post it here, if anyone want me to...)

        The difference is probably crucial. Sodium acetate is just a salt, but guanidium salts are powerful denaturants.

        --
        Phillip
        Attached Files

        Comment


        • #5
          I'm sorry I described inappropriately, it's not a peak at 230, it's just 260/230 is very low.

          Originally posted by ECO View Post
          It's the guanidium-SCN in the QG. An extra (or even a third) PE wash can help. In my experience it can affect efficiencies of sensitive enzymatic reactions, but is not a huge problem for amplification based preps.

          Comment


          • #6
            In my experience, this type of curve is not unusual to see with the gel extraction kit. The moderator is correct, you should introduce more washes with PE than the manual states. This will help mitigate this peak, however it probably will not get rid of it completely. Normally, I would usually follow-up with some kind of buffer exchange (Ampure beads, Microcon, or drop dialysis), but I was always concerned about losing more sample and potentially introducing more bias. Thus, I would usually go with drop dialysis. Not sure many people do that anymore or are comfortable floating their sample on a dialysis disk in a bin of water.

            Comment


            • #7
              Originally posted by skblazer View Post
              I' sorry, it's the peak shorter, it's just significantly higher at 230 than 260. (please see attached)

              So the sodium acetate should not be a problem, am I right?
              That looks like sodium acetate to me. A good paper on the UV spectrum of aqueous acetate is this one:

              Reference Type: Journal Article
              Author: Ruderman, Graciela
              Author: Caffarena, Ernesto R.
              Author: Mogilner, Inés G.
              Author: Tolosa, Eduardo J.
              Primary Title: Hydrogen Bonding of Carboxylic Acids in Aqueous Solutions—UV Spectroscopy, Viscosity, and Molecular Simulation of Acetic Acid
              Journal Name: Journal of Solution Chemistry
              Cover Date: 1998-10-21
              Publisher: Springer Netherlands
              Issn: 0095-9782
              Subject: Chemistry and Materials Science
              Start Page: 935
              End Page: 948
              Volume: 27
              Issue: 10
              Url: http://dx.doi.org/10.1023/A:1022615329598
              Doi: 10.1023/A:1022615329598

              Is it a problem? Well to be able to see acetate on a nanodrop you probably need to be at 100 mM or more. It is not as bad as having guanidium salts in your sample for most downstream applications. But it can definitely cause problems to have 100 mM salt in your sample.

              --
              Phillip

              Comment


              • #8
                Thanks for the advice. I'll try washing twice by PE.

                Originally posted by Cofactor Genomics View Post
                In my experience, this type of curve is not unusual to see with the gel extraction kit. The moderator is correct, you should introduce more washes with PE than the manual states. This will help mitigate this peak, however it probably will not get rid of it completely. Normally, I would usually follow-up with some kind of buffer exchange (Ampure beads, Microcon, or drop dialysis), but I was always concerned about losing more sample and potentially introducing more bias. Thus, I would usually go with drop dialysis. Not sure many people do that anymore or are comfortable floating their sample on a dialysis disk in a bin of water.

                Comment


                • #9
                  Hi Phillip,

                  Many thanks for the paper.

                  But do you know where the huge sodium acetate come from? Is it from buffer QG to buffer the PH? But I didn't hear a lot of people report this. I'll try wash the columns twice by PE.

                  Originally posted by pmiguel View Post
                  That looks like sodium acetate to me. A good paper on the UV spectrum of aqueous acetate is this one:

                  Reference Type: Journal Article
                  Author: Ruderman, Graciela
                  Author: Caffarena, Ernesto R.
                  Author: Mogilner, Inés G.
                  Author: Tolosa, Eduardo J.
                  Primary Title: Hydrogen Bonding of Carboxylic Acids in Aqueous Solutions—UV Spectroscopy, Viscosity, and Molecular Simulation of Acetic Acid
                  Journal Name: Journal of Solution Chemistry
                  Cover Date: 1998-10-21
                  Publisher: Springer Netherlands
                  Issn: 0095-9782
                  Subject: Chemistry and Materials Science
                  Start Page: 935
                  End Page: 948
                  Volume: 27
                  Issue: 10
                  Url: http://dx.doi.org/10.1023/A:1022615329598
                  Doi: 10.1023/A:1022615329598

                  Is it a problem? Well to be able to see acetate on a nanodrop you probably need to be at 100 mM or more. It is not as bad as having guanidium salts in your sample for most downstream applications. But it can definitely cause problems to have 100 mM salt in your sample.

                  --
                  Phillip

                  Comment


                  • #10
                    Hi, I recently just saw what you have seen.

                    The first image is from a nanodrop: Comparison of what a 1 wash PE looks like compared to straight qiaquick purification w/o gel elution.

                    2nd image: parallel amplicon, except this time 5x wash through a vacuum manifold. I really think its the Buffer QG that is effecting this. I'm not sure how to completely get rid of it, but the multiple washes does seem to remove it somewhat. I'm going to try to sequence it and see if the quality is sufficient.

                    Please let me know if you've found a way of purifying it out completely. Thanks!
                    Attached Files
                    Last edited by aureias; 12-07-2011, 09:03 AM.

                    Comment


                    • #11
                      If it is really bothering you, just dilute the sample to ~500ul and concentrate it with a microcon spin column. Re-dilute a few times (on the same column), if necessary. That will get rid of acetate and/or guanidium.

                      But if you are getting a large peak like that, there is probably a technique issue. You might want to call Qiagen and ask for advice on how to avoid the carry over.

                      --
                      Phillip

                      Comment


                      • #12
                        Originally posted by aureias View Post
                        Hi, I recently just saw what you have seen.

                        The first image is from a nanodrop: Comparison of what a 1 wash PE looks like compared to straight qiaquick purification w/o gel elution.

                        This is what a 5x wash through a vacuum manifold looks like of a gel fragment. I really think its the Buffer QG that is effecting this. I'm not sure how to completely get rid of it, but the multiple washes does seem to remove it somewhat. I'm going to try to sequence it and see if the quality is sufficient.
                        Wait, I just re-read this. What are samples on the two runs? On the first picture I see a green peak centered around 230 nm, and a black peak around 260 nm. Which is which?

                        --
                        Phillip

                        Comment


                        • #13
                          ahh sorry.

                          first image is a comparison of PCR amplicon purifications with either a pcr purification qiaquick or else a pcr gel purification qiaquick using standard recommended protocol.

                          Qiaquick Gel Purification (green line) (buffer QG)
                          other lines: Qiaquick PCR purification without gel extraction (buffer PB)

                          second image was a parallel gel purification with 5x washes. It cleared up some of the peak, but still not as clean as I'd like.
                          Last edited by aureias; 12-07-2011, 09:02 AM.

                          Comment


                          • #14
                            Hmm, gave them a call. As expected, its the salts from QG.

                            A few things they said might improve it is:

                            longer incubations in the wash buffer
                            incubations at 37 degrees in the wash buffer
                            multiple washes

                            I've done multiple washes, and that seems to eliminate it a fair bit, not completely, but somewhat.

                            I'm going to give the other options a try and will see what pops out.

                            Comment


                            • #15
                              When we see that 230 nm peak (guanidine isothiocyanate), we just do a microcon. But we use those spin filters moderately frequently, so they are available.

                              --
                              Phillip

                              Comment

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