Upper limit of MgCl2 in PCR
Taek H. You
tyou at postbox.acs.ohio-state.edu
Wed Jun 11 02:32:09 EST 1997
>Ng Hian Cheong (medp3019 at leonis.nus.sg) wrote:
>What is the reason for the existence of an upper limit of MgCl2
>concentration during PCRs ?? What is this value, 10 mm or 15 mM ?
>I have experience this "upper limit of MgCl2 conc. in PCRs b4:
> I was studying a family of genes by PCR amplifying their genes
>segments from genomic DNA. At 1.5 mM, I identified the first gene. At 3.0
>mM, the second gene together with the first gene were amplified. At 4.5,
>6.0 & 7.5 mM, only the second gene segmemt was amplified. It was also
>noticed that PCR product at 7.5 mM was lesser in amount than those at 6
>mm as judged from intensity of their bands in agarose gels. At 9.0 mM
>MgCl2, NO PRODUCTS was observed. What is the reason for this absence of
>PCR products at high MgCl2 conc. ? Any suggestions. You are welcome to
>reply directly to medp3019 at leonis.nus.sg.
>Thousand of thanks
I also have the similar experience with a multigene family. But, I could not
find exactly what make it happen.
Here is general roles of Mg++ ion in PCR.
Mg++ is correlated with DNA template
dissociation, primer annealing, and nucleotide availability.
As Mg++ increases, ds DNA may require more heat for denaturation while
primers can anneal more stably (in a sense, to nonspecific sites too). More
importantly, Mg++ binds to nucleotides. Polymerization requires free
nucleotides, not Mg++-bound nucleotides to synthesize. This can be overcome
with higher nucleotide concentration.
Therefore, it might be one or more of the factors in your case.
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