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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