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  • dilution help

    I need help with this problem. You don't need to know the science, it is more of a dilution and concentration math problem.

    I have a standard reaction setup below with these reagents:

    H20 = 32.75ul
    10x PCR buffer = 5ul ---> final 1x
    MgCl2 (25mM) = 3ul -----> final 1.5mM
    Primer F (10uM) = 2.5ul ---> final 0.5uM
    Primer R (10uM) = 2.5ul -----> final 0.5uM
    dNTP (10mM) = 1ul ------>final 0.2mM
    DMSO (100%) = 1ul ------>final 2%
    DNA = x
    Taq polymerase (5U) = 0.25ul ----> final 0.025U
    Total = 50ul

    The DNA must be 1ug in 50ul. I have DNA at 20ng/ul.

    The only way can get 1ug in 50ul is if I add 50ul of DNA into the reaction. But even if I make H2O = 0ul, I will still be about 15ul over my required 50ul, and the final concentrations of each reagent will change.

    I need to have 1ug of DNA in the reaction but keep my final reagent concentrations the same (or close as possible) to the standard reaction above. I am sure I can find a way if I keep plugging away with the numbers but I don't have that much time. Can someone help me?

  • #2
    Re: dilution help

    Originally posted by Unregistered View Post
    I need help with this problem. You don't need to know the science, it is more of a dilution and concentration math problem.

    I have a standard reaction setup below with these reagents:

    H20 = 32.75ul
    10x PCR buffer = 5ul ---> final 1x
    MgCl2 (25mM) = 3ul -----> final 1.5mM
    Primer F (10uM) = 2.5ul ---> final 0.5uM
    Primer R (10uM) = 2.5ul -----> final 0.5uM
    dNTP (10mM) = 1ul ------>final 0.2mM
    DMSO (100%) = 1ul ------>final 2%
    DNA = x
    Taq polymerase (5U) = 0.25ul ----> final 0.025U
    Total = 50ul

    The DNA must be 1ug in 50ul. I have DNA at 20ng/ul.

    The only way can get 1ug in 50ul is if I add 50ul of DNA into the reaction. But even if I make H2O = 0ul, I will still be about 15ul over my required 50ul, and the final concentrations of each reagent will change.

    I need to have 1ug of DNA in the reaction but keep my final reagent concentrations the same (or close as possible) to the standard reaction above. I am sure I can find a way if I keep plugging away with the numbers but I don't have that much time. Can someone help me?
    You can't get around it. 1g = 1000ng.
    In order to get 1g of DNA, you need 50L of 20ng/L solution.

    Therefore, you need a more concentrated solution to start with, anything you add to it will just be diluting the DNA concentration.

    Comment


    • #3
      Re: dilution help

      Hello,

      Sorry not to be of any help to your question. But I am having a slight issue with diluting concentrations. I need to use 2mM dNTP in my PCR reactions at a volume of 5 micro litres per sample. Can anyone help me with how I can obtain a 2mM dNTP concentration??

      Many thanks for your advice.

      Abbi

      Comment


      • #4
        Re: dilution help

        Originally posted by Unregistered View Post
        Hello,

        Sorry not to be of any help to your question. But I am having a slight issue with diluting concentrations. I need to use 2mM dNTP in my PCR reactions at a volume of 5 micro litres per sample. Can anyone help me with how I can obtain a 2mM dNTP concentration??

        Many thanks for your advice.

        Abbi
        Hi Abbi, what concentration do you have to start with? 2mM means 2 millimoles per litre.

        5L will need 10 nanomoles of your dNTP

        (1mL has 2moles, 1L has 2nanomoles.)

        Comment

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