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  • #16
    Reviving a slightly old thread but this same problem happened to me (unfortunately before I read this). Is there a way I can further clean my samples that are already gel purified via Qiaquick? Can I apply the eluted samples back on a Qiaquick column and perform the extra washes (with incubation) as suggested here? Or does anyone have another other suggestions on how to remove the salts post Qiaquick?

    Thanks in advance for any help!

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    • #17
      Geeting high absorbance at 230 nm for DNA extract

      Hi all,

      Very productive discussion indeed. I am experiencing a similar problem. I did DNA extraction from water samples using phenol/chloroform. However, I do get a shoulder/high peak at 230 nm whereas there is a slight peak at 260 nm. My 230:260:280 ratio is more like this: 1.6:0.8:0.5.

      I adapted my fieldwork sampling protocol from Foote et al. (2012)*. Based on the literature search, and similar problems people have experienced, I do think that the high peak at 230 nm is due to some salt contamination along with residual ethanol. I am in the midst of troubleshooting, but I am not sure how to fix this problem. There are people suggesting to use ether (to remove organic solvents) or to dialyse the probe if salt is the contaminant. Also, during PCR some additional reagents can be used, such as BSA, glycerol to prevent any potential inhibitors getting amplified.

      Any suggestions on how to carry out? I would like to have purified DNA before troubleshooting with PCR step, ideally.

      *http://www.plosone.org/article/info%...l.pone.0041781
      Last edited by Bilgenur; 10-23-2014, 07:16 PM.

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      • #18
        Using 1.8x Ampure or similar beads are good options and amenable to high throughput methods. If you have high molecular weight DNA, you can elute with warm buffer with longer incubation to minimise loss.

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        • #19
          Originally posted by nucacidhunter View Post
          Using 1.8x Ampure or similar beads are good options and amenable to high throughput methods. If you have high molecular weight DNA, you can elute with warm buffer with longer incubation to minimise loss.
          Agree.
          Look, the problem here isn't that the poster doesn't have a desirable A230:A260 ratio. The problem is that the poster's prep methodology left contaminating salts mixed in with their "purified" sample. Some of these salts have a UV spectrum that you see when you look. Chances are that if you had assays that could detect non-optically active substances, you would see lots of stuff there as well.
          For example see this post.
          Does it matter? Who knows. Generally a bunch of optically active substances showing up in your sample isn't a desired outcome, but there are simply too many factors involved to say.
          So my tendency is to say stop using UV spec. Use a Qubit instead.

          --
          Phillip

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          • #20
            I am facing a very similar problem to Bilgenur. We are doing DNA extraction from water samples using phenol/chloroform, and we get a (very) high peak at ~230 nm. We also have sometimes a smaller peak at ~270 which is probably phenol. The pellet we see after ethanol precipitation is large and greyish to white, and I suspect it is mostly salts rather than DNA.

            Originally posted by nucacidhunter View Post
            Using 1.8x Ampure or similar beads are good options and amenable to high throughput methods. If you have high molecular weight DNA, you can elute with warm buffer with longer incubation to minimise loss.
            I did try a 1.8x AMpure cleanup, but for some reason we lost all the DNA. I speculate that the contaminants in the extracted DNA (phenol, salts,..?) interfered with the properties of the AMpure buffer and prevented the binding of DNA to the beads. Or did I overdry the beads (~2 min)?

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            • #21
              Your speculation is plausible, but I would consider follwoing:
              1- lower than 70% concentration of ethanol used for washes
              2- faulty beads (can you try another sample with those beads?)
              3- 2 min for drying often is not enough. Good indications are loss of gloss and cracks just appearing
              4- Volume of beads used. It is good practice to dilute samples to minimise effect of potential contaminants. So it is better to use 90 ul of bead with 50 ul of DNA rather than 18 ul with 10 ul of DNA

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              • #22
                Originally posted by nucacidhunter View Post
                Your speculation is plausible, but I would consider follwoing:
                1- lower than 70% concentration of ethanol used for washes
                2- faulty beads (can you try another sample with those beads?)
                3- 2 min for drying often is not enough. Good indications are loss of gloss and cracks just appearing
                4- Volume of beads used. It is good practice to dilute samples to minimise effect of potential contaminants. So it is better to use 90 ul of bead with 50 ul of DNA rather than 18 ul with 10 ul of DNA
                Thanks for the quick reply!
                1. We used fresh 80 % for the washes.
                2. We have used these beads many times with no problems (but not for genomic DNA).
                3. Could be the problem, but seems unlikely.
                4. Agreed, that was my thought as well.

                Many people in my lab believe that Ampure beads will not work with genomic, high molecular weight DNA. So now I am not sure if I should try again with Ampure beads or try a column purification, or play with the protocol and do multiple washes with ethanol.

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                • #23
                  I have cleaned HMW DNA with AMPure beads many times with good results. I incubate bead-DNA mix for 10 min and elute with warm buffer for 5 min. Once I had a sample extracted with phenol and addition of beads caused unusual cloudy appearence but result was fine. If you are going to use columns watch for blockage. Viscous material can block them, again dilution can help.

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                  • #24
                    Originally posted by nucacidhunter View Post
                    I have cleaned HMW DNA with AMPure beads many times with good results. I incubate bead-DNA mix for 10 min and elute with warm buffer for 5 min. Once I had a sample extracted with phenol and addition of beads caused unusual cloudy appearence but result was fine. If you are going to use columns watch for blockage. Viscous material can block them, again dilution can help.
                    Thanks again! How warm is the buffer?

                    Comment


                    • #25
                      37C and incubate at that temperature. Occasionally I have eluted at 56C as well when DNA quantity was low to maximise recovery.

                      Comment

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