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Thread: PCR failures :(

  1. #1 PCR failures :( 
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    Hi I am a honours student starting only this year. I have done PCR about 3 years ago for about 2 months. The reactions then had worked well. I had +1 reaction volume to all my master mix to reduce pippetting error and also to prevent running out of Master Mix for the last tube. I never had problems with the pippetting.

    Now I have started new work on my honours project and has been having PCR failures for at least 10 reactions.

    For the first few reactions, we thought it was pippetting error because I was new. Then after that I changed the primers. I designed and bought new ones. I have tried a few of them and the result is still the same. My supervisor disagrees to my +1 method and insist that I need to work on my pippetting technique. It was alright sometimes and more often than not, I don't end up with the correct volume of leftovers and sometimes run out of MM for the last tube.

    Is pippetting error really detrimental to a PCR reaction or is it really just my primers?


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  3. #2  
    Moderator Moderator Cogito Ergo Sum's Avatar
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    There are many things that can go wrong whilst performing a PCR: the annealing temperature can either be too high or too low, the difference between the melting temperatures of your primers is more than 5C, the primers form secondary structures or show intramolecular base-pairing, etc.

    Nonetheless, you can check the quality of your primers with primer3plus or BLAST.

    Yet, pipetting errors can be detrimental, because a PCR mixture is max. 50 l. Small volumes are more difficult to work with and cause relatively great errors.
    Thus, I do think that your pipetting is the cause of your problems, but I would also double-check your primers if I were you.


    PS: Have you tried a Touchdown PCR?


    "The only safe rule is to dispute only with those of your acquaintance of whom you know that they possess sufficient intelligence and self-respect not to advance absurdities; to appeal to reason and not to authority, and to listen to reason and yield to it; and, finally, to be willing to accept reason even from an opponent, and to be just enough to bear being proved to be in the wrong."

    ~ Arthur Schopenhauer, The Art of Being Right: 38 Ways to Win an Argument (1831), Stratagem XXXVIII.
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  4. #3  
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    Hi! Thanks for your help! The PCR run after this message worked! Just when I was getting desperate..

    Yes, it was the primer. I had checked the quality of my designed primer using NetPrimer.

    I should refine my pippetting skills and yes all my PCR runs so far has been a Touchdown PCR because I am still trying out which primers could work. Once I have working primers, my number of samples each cycle would be larger and thereby reducing the pippetting error.

    Thanks again!
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  5. #4  
    Moderator Moderator Cogito Ergo Sum's Avatar
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    Quote Originally Posted by ahbu View Post
    Hi! Thanks for your help! The PCR run after this message worked! Just when I was getting desperate..

    Yes, it was the primer. I had checked the quality of my designed primer using NetPrimer.

    I should refine my pippetting skills and yes all my PCR runs so far has been a Touchdown PCR because I am still trying out which primers could work. Once I have working primers, my number of samples each cycle would be larger and thereby reducing the pippetting error.

    Thanks again!
    You're welcome.
    "The only safe rule is to dispute only with those of your acquaintance of whom you know that they possess sufficient intelligence and self-respect not to advance absurdities; to appeal to reason and not to authority, and to listen to reason and yield to it; and, finally, to be willing to accept reason even from an opponent, and to be just enough to bear being proved to be in the wrong."

    ~ Arthur Schopenhauer, The Art of Being Right: 38 Ways to Win an Argument (1831), Stratagem XXXVIII.
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  6. #5  
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    I am having problems with my PCR again. This time with the negative lane.

    I had a contamination before (there are a specific product in the negative lane) and I cleaned it out. New reagents, cleaned pipettes etc. Then I tried a new PCR again with micro satellite primers and they worked beautifully for a while.

    Then for the last 2 reactions (using the same reagents and procedures after the contamination), I have multiple low molecular weight bands in the negative and no products at all. The bands actually look like a ladder and more prominent than the ladder itself! Could it be that the contamination wasn't cleared?

    Btw, I ran a touch-down PCR.
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