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PCR additives

  1. Mar 26, 2003 #1

    Monique

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    OK, let's get some molecular biology in here. I would really like to know exactly how many PCR additives there are and why they would improve the reaction.

    I am talking about reagents such as DMSO, Tween, Formamide, Glycerol, spermazin?, (NH4)2SO4 etc.


    for those who don't know: PCR (polymerase chain reaction) is a technique that linearily or exponentially amplifies DNA, such as specific genes or 'just' the whole genome.
     
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  3. Mar 27, 2003 #2

    Another God

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    Wow!

    There are PCR additives? Why? Seems to work fine without them doesn't it?

    I did PCR today, and we used the usuall stuff (all of the nucleotides, Forawrd primer, reverse primer, Taq DNA polymerase..)

    Oh, there they are...
    we used Tris HCL pH 8.8, MgCl2 and..... here it is : (NH4)2SO4

    I ahve no idea what those three additives do. Obviously, MgCl2 is a salt, it probably does some salt binding thing with the DNA....but to what effect I don't know.

    Thanks for posting this, I will ahve to ask why they are in there now.
     
  4. Mar 27, 2003 #3

    Another God

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    Would the Glycerol count as an oil? I know we added oil to stop the water from evaporating as it is heated through the cycles.
     
  5. Mar 27, 2003 #4

    Monique

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    There sure are And no, PCRs don't always work without them.. I have run into this problem the hard way. It is true that the standars PCR consists of the ingredients you describe.. but sometimes the primers are unstable on the sequence or the sequence is rich in secondary structures, that is when the additives come in.

    Oh! I have to attend an seminar.. be back later.
     
  6. Mar 31, 2003 #5
    Additives such as glycerol, DMSO, betaine are very effective (you need luck as well), when your DNA has a high GC region. MgSO4 can be replacing MgCl2 when your GC porcentage is high due to it helps to destabilize secondary structures in DNA.
    There should be more additives....let me think
     
  7. Apr 2, 2003 #6
    DMSO and formamide (N,N'-dimethylformamide, right?) are polar solvents, they'd help solvate denatured DNA. So would glycerol. Tween and spermazine? Never heard of them. Is the last a polyamine like spermidine? With a hydrazine linkage? Ammonium sulfate I imagine would just be a salting in efffect.
     
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