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    • #15193
      melis
      Participant

      Hello,

      My problem is about an interesting smearing of my PCR product. I have done PCR of a plant nuclear DNA with about 6 kb many many times with my specific and well performing primer. About 200 samples of PCR products were obtained without any problem during 3 months i.e. I got sharp PCR bands during this 3-month-period. The sequencing reactions were also very well. However, about for a week, my PCR products have started to give full smear image. I made optimization for my PCR several times by changing all the reagents (dNTP, MgCl2, primer, Taq, Buffer, template and dH2O). I’ve also isolated the DNA’s once more considering any probable degradation of my templates and sterilize all my equipments (including pipetes and tips) but still got the same result.

      My questions is although I’ve changed all the reagents and made optimizations several times, what may be the cause of this suprising result though I’ve got very sharp bands hundreds of times until a week ago? The ultimate reason for this situation may be the degradation of my previously well-perfoming stock primer and unfortunately could not find any other 😥 Am I right or could it be some other reasons? Please help!!!

      Thanks

      Melisa

    • #105639
      canalon
      Participant

      Can you reorder fresh stock? It is cheap and fast generally…
      Sterilizing pipette is of dubious use, cleaning them with a mild acid (0.1M HCl for 10 minutes) is a better way to degrade potential contaminants. But that is more in case of contamination than smearing. Also Thermocycler can go bad, do you have a way to check that your block is heating properly (temperature probe, other PCR working?)

    • #105648
      melis
      Participant

      Thanks,

      I reordered all the reagents except primers. So, I finally suspect my primers. After fresh primer usage, if I face with the same problem, I dont know what to do 😥

      I sterilized my pipettes with UV and tips with autoclave. but I did not do anything with HCl. I shall try it.

      I have 4 thermocyclers. Most of the time I have used 2 of them. May be the problem is related with them but my labmates also use same thermocyclers but they dont have the same problem 😥

      Still I hopelessly try to find out the problem.

      Thanks by the way

      Melisa

    • #105649
      canalon
      Participant

      HCl (or light acid treatment) is to remove DNA contamination from the inside of the "nose" of the pipette. It is best to dismantle the pipette and treat only the pieces that can produce aerosols (the nose and the piston inside), and avoid treating the metal part. A 10 to 15 minute path, followed by distilled water rinsing, and 90% EtOH for quick drying works wonder.

      This is what I do if I start seeing amplification in my negative controls, and it generally works well, But I am not sure if this is going to be useful for smearing.
      The thermoccyler thing is a bit of a long shot (they tend to work well, or not at all), and if your colleagues have no problem, I would say that it probably rules them out as potential culprit.

    • #105835
      audacity92
      Participant

      Did you ever figure out what caused the smearing? I am having the exact same problem right now – 150 PCR reactions with the same primers and suddenly whole lane smearing. I’ve changed everything, ordered new primers, even used different pipettes in a different location and I still see the smearing! I even went back to DNA that had worked previously and now I get smears for those as well. I would love to know what is causing it! Any insight? Thanks!

    • #105843
      jonmoulton
      Participant

      Have y’all run a control on your electrophoresis setup? Be sure to run a commercial size-standard DNA ladder on the gel and see whether it smears too. If so, your PCR reagents and procedure may be fine — it’s time to check the electrophoresis buffers, matrix and physical setup. If the ladder looks sharp, then your attention should be on the PCR steps.

    • #105844
      dinofernando
      Participant

      Have you tried double checking your program setup on your thermocycler?

      Might have accidentally changed it?

    • #105858
      Cat
      Participant

      I encountered similar problem at times and found the culprit to be the loading dye and/or electrophoresis buffer…

    • #115298
      priyah
      Participant

      hello all ,
      Even i have a similar problem. I am using 3 different primers to amplify 3 different exons of a gene . 1 of the exon is amplifying very well ( a single bright band ) but the other two exon amplification is not coming properly.and this has occured recently. ( even though the PCR reaction was standardized earlier ) I have used same reagents , pipettes , PCR machine , reconstituted new primers for amplifying all 3 exons . Interestingly sometimes I do get a single bright band , but this happens for 1 in 10 samples and rest appear as smear . I checked the DNA quality and its good ,,..Really I dont know what to do next .. plz suggest.

      Thanks.

    • #115299
      JackBean
      Participant

      Just a stupid idea, but if you digest your DNA with some RE cutting between your primers, do you get rid of the smear?

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