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Thread: PCR/SDS-PAGE Post Mortem

  1. #1 PCR/SDS-PAGE Post Mortem 
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    Hi,

    I carried out PCR on a polynucleotide of interest recently and then used SDS-PAGE to analyse its migration. At least that was the aim. What happened in reality is I saw no bands except those for the DNA marker, suggesting that I did not extract enough DNA from the plant leaves I was using? Furthermore, the gel itself looks sort of smeary and way, way paler than normal. It was suggested that I may have used too much agarose, yet I weighed out the mass to the exact specified amount.

    Please if anyone can help me and tell me what I may be doing wrong that would be great. I would really like to get a handle on PCR & SDS-PAGE!

    A sad Tridimity


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  3. #2  
    Forum Cosmic Wizard paralith's Avatar
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    I'm not as familiar with SDS PAGE, but there are many ways in which PCR can go wrong. I would first just try the PCR again without changing anything, and see what happens. If you get the same result, then you start trying to figure out what may have gone wrong with the PCR. In some cases long smears can actually be indicative of too much sample DNA being added to the original reaction, so try the PCR again but add less of your DNA and more water. (Or else dilute your DNA samples separately.) Have you checked the concentration of your DNA? It's usually optimal to have a concentration of around 100 ng/ul.


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    Forum Cosmic Wizard spuriousmonkey's Avatar
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    1. use a POSITIVE control. If not, you can repeat the experiment a zillion times and still not know anything. That is then a PCR reaction with primers and template under the same program that has proven to work!

    2. SDS page is used for proteins, not DNA. Use a agarose gel stained with ethidium bromide. Unless your insert is really really tiny, then you can use other stuff..You can see your bands under a UV light.

    Anyway. Give some more info. Size of expected insert. Program. TMs, your mix. and that kind of shite.
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  5. #4  
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    Just from how you worded the question (so dont be offended if you did), did you run DNA quantifications before you ran the PCR?

    I had uge problems in a recent project with my PCRs. Long story short I had to change my primers and mix: is it possible these havent been maintained correctly?
    Have you considered altering the magnesium?

    Sorry if this isnt relevant, I dont know the SDS page protocol.
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  6. #5 Re: PCR/SDS-PAGE Post Mortem 
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    Quote Originally Posted by tridimity
    Hi,

    I carried out PCR on a polynucleotide of interest recently and then used SDS-PAGE to analyse its migration. At least that was the aim. What happened in reality is I saw no bands except those for the DNA marker, suggesting that I did not extract enough DNA from the plant leaves I was using?
    Did you spec your isolates for DNA?

    Edit: Read the other answers Bio... everyone else asked that question already!
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  7. #6 Thanks guys.. 
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    Thankyou all for replying.

    Unfortunately, the procedure was a scheduled group activity in a multiuser lab, so I will be unable to repeat the experiment anytime soon. And it is my mistake, we did not use sodium dodecyl sulphate, it was just standard agarose gel electrophoresis. As for checking the concentration of DNA, we were basically encouraged to grind the leaves as much as possible, greener samples apparently being better! To be honest, it is not a fault with the procedure itself that is the problem - several other groups carried out exactly the same procedure, and got the desired result. So it will be some practical mistake I am making.

    More information about the procedure...

    We were given four plants - Arabidopsis thaliana. Two of the plants were parent plants; one (Landsberg) homozygous FUF/FUF (wild tpye), one (Colombia) fuf/fuf mutant.
    Other 2 plants of unknown genotype. The overall aim of the procedure was to identify the genotype of the unknowns.
    We were trying to look at the segregation of microsatellite markers, in this case using an AT repeat.

    PCR mix:

    KCl 50mM
    Tris-HCl (pH 9) 10mM
    Triton X-100 0.1%
    MgCl2 1.5mM
    dNTPs 0.2mM
    Oligo 1 20pM
    Oligo2 20pM
    TAQ polymerase 0.6 units

    Programmed cycles:

    (94C, 3 min) x1
    (94C, 15s - 55C 15s - 72C 30s) x35
    (72C, 2 min) x1

    So.. any ideas what I might have done wrong?


    Thanks again,

    Tridimity
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  8. #7  
    Moderator Moderator TheBiologista's Avatar
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    Right well the rigid protocol and the fact that it did work for some pretty much suggests you made a mistake somewhere. From your description of the gel there are a few possibilities I can think of.

    1. Your DNA isolation went askew- can you tell us more about the protocol for that part? Did you use solvents, some kind of phase separation? I don't tend to do DNA isolation myself (when I do, I use a Qiagen kit)- but I know from doing mRNA isolations that it can be easy to use the wrong solvent at the wrong stage, or discard the wrong solvent layer and thus lose your isolate, perhaps without realising.

    2. How careful were you with your Taq? Typically very small volumes are used in most PCR master mixes. When you take 1-2ul up in a gilson it can often be pretty hard to see in the tip. I always make a point of carefully checking the tip whenever I take up a small volume and I'm always careful to watch when I deposit it too. A 2ul bubble looks a lot like a 2ul volume of taq!

    3. Least likely problem: The pale and "smeary" gel may indicate that your % agarose was off. If by pale, you mean transparent, then you used too little agarose. If pale means white, then possibly too much. In the latter case, you'd still expect to see bands but you expect them to have migrated a shorter distance. But since you can see the DNA marker, a ladder I assume, you can be pretty sure that the gel was fine, even if it wasn't perfect.
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  9. #8  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    Maybe you had too much DNA to start with.
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  10. #9  
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    What did the bands from your marker look like? If they are clear and distinct, then the problem is for sure not with your gel.

    From your descriptions, I'd say chances are the problem lies with your template DNA. I work with Arabidopsis and have done quite of bit of mapping in the past, which translates into literally thousands of PCRs on markers of all types. Usually if I didn't get a product, it was because of some problem with the template, i.e. the genomic DNA.

    Which protocol did you use to isolate the DNA? I hope it wasn't with a kit, I'm a bit old school in those regards and hate kits. Anyhow, with plant genomic DNA, the two primary ways are the CTAB and Dellaporta protocols.

    Assuming you used one of these protocols, you should have been able to see a DNA pellet in the last steps. If you didn't, then you may not have any DNA. The other problem is that if you did it wrong you may have sheared the DNA.

    Did you try quantifying your template at all prior to see if you even had any genomic DNA?

    The other thing is that when doing a PCR from genomic DNA, be kind of liberal with the amount you use for template.
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  11. #10  
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    Run 5 ul of the isolated DNA on a 0.7% agarose gel to confirm you do have DNA. And you do need to quantify the DNA after isolation. Too much template is as bad as too little.

    Use an optimum amount of the template [between 50 and 100 ng/ul] by diluting the stock DNA is necessary for the PCR. Use a negative control [PCR water instead of DNA] and a positive control [DNA standard]. That will show you if the PCR was done properly. Then run it on 1.2-1.5% agarose gel to check the PCR product.
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  12. #11  
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    Quote Originally Posted by samcdkey
    Run 5 ul of the isolated DNA on a 0.7% agarose gel to confirm you do have DNA. And you do need to quantify the DNA after isolation. Too much template is as bad as too little.

    Use an optimum amount of the template [between 50 and 100 ng/ul] by diluting the stock DNA is necessary for the PCR. Use a negative control [PCR water instead of DNA] and a positive control [DNA standard]. That will show you if the PCR was done properly. Then run it on 1.2-1.5% agarose gel to check the PCR product.
    He can't re-run the experiment. I reckon he was looking for some probable explanations to put in his write-up.
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  13. #12  
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    Quote Originally Posted by TheBiologista
    He can't re-run the experiment. I reckon he was looking for some probable explanations to put in his write-up.
    If he has the stock DNA he could still quantify it. If the DNA conc is high, then too much template could be a good dodge. If there is no DNA that will be an even better explanation! :P
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  14. #13  
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    Quote Originally Posted by samcdkey
    Quote Originally Posted by TheBiologista
    He can't re-run the experiment. I reckon he was looking for some probable explanations to put in his write-up.
    If he has the stock DNA he could still quantify it. If the DNA conc is high, then too much template could be a good dodge. If there is no DNA that will be an even better explanation! :P
    Yeah but I gather it was an undergraduate lab. He doesn't have lab access, let alone access to the sample, which I'm sure was probably just discarded at the end of the practical.
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