Mastermix in PCR

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    • #16882
      klee5533
      Participant

      Hey guys,

      I ran PRC on some genes yesterday, as a college student in a research lab, and I feel like I messed up the amount of mastermix solution I put into my PCR samples. So I made 10 PCR tubes, each of them including sdWater, different fwd primer and rev primer, DNA template, and mastermix in total volume of 50 microliter. My mastermix includes 10x butter, dNTPs, MgCl2, and Taq Polymerase, and each of the tube needed to have 13.25 microliter of mastermix solution. But I think I put around 14.5 microliter… Yes, that was stupid of me. I am going to let my professor know tomorrow since I realized my mistake, but I just wanted to know what’s the biological cause of the excessive amount of mastersolution on PCR run going to be. Would it do anything else than maybe promoting the reaction further because the concentration is higher? I cannot think of something since I am not that familiar with PCR work!

      Please help me out by sharing you knowledge! I really appreciate your time, guys!

    • #112537
      JackBean
      Participant

      If it was 1.25 ul in 50 ul total, I wouldn’t be much concerned. However, I would probably not compare these results to others with standard set-up.

    • #112693
      daddyjames
      Participant

      I agree with what JackBean said, however it does not answer your question.

      MgCl2 is a required cofactor for the Taq Polymerase to work. The activity of the Taq polymerase is sensitive to the concentration of MgCl2. The higher the concentration of the MgCl2, the more active the enzyme is. As the concentration of MgCl2 increases, the incorporation of dNTPs increases. However, it also increases the the error rate – the rate at which an incorrect dNTP is incorporated into the resulting PCR product as compared with the original sequence.

      MgCl2 also can stabilize dsDNA structures. Manipulation of MgCl2 concentration (increasing it) is often used to increase amplification of "hard to amplify" DNA samples. However, this increases the amplification of "non-specific" PCR products. PCR products that are formed from the stabilization of the PCR primers binding to slightly different DNA sequences – which would not occur under lesser concentrations of MgCl2.

    • #113204
      stonjohn4
      Participant

      Hi All,
      I am having the following problem. I work with a fusion gene and need to do a QPCR. I found a couple of papers with primers I can use but have no idea how people manage to design those primers and what is the product length etc.

    • #113217
      JackBean
      Participant

      for qPCR your primers should have very similar Tm and be pretty close (around 100-150 bp)

    • #115606
      Johni
      Participant

      This is such a pleasant conventional and great post….you shook posting it much appreciated a lot….!!!!

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