PCR amplification effects by the MgCl2 concentration. That what happen in an experiments I carried out. I need more advices and recommendation until what concentration the amplification could yield good result in the PCR
Hi Aldosky, Magnesium chloride, act as source of magnesium ion for PCR, influence the primer template annealing temperature, fidelity, specificity, and yield. Standard PCR protocols need final magnesium ion concentration of 1.5 mM. The optimum magnesium concentration for Taq DNA polymerase is generally between 1-4 mM in the presence of an overall dNTP concentration of 0.8 mM. Excess magnesium can cause an increase in nonspecific product, while too little can cause reduced yield. The presence of EDTA and other chelating agents, often found in template DNA, can affect the concentration of free magnesium in the PCR
regards
Hi Aldosky, Magnesium chloride, act as source of magnesium ion for PCR, influence the primer template annealing temperature, fidelity, specificity, and yield. Standard PCR protocols need final magnesium ion concentration of 1.5 mM. The optimum magnesium concentration for Taq DNA polymerase is generally between 1-4 mM in the presence of an overall dNTP concentration of 0.8 mM. Excess magnesium can cause an increase in nonspecific product, while too little can cause reduced yield. The presence of EDTA and other chelating agents, often found in template DNA, can affect the concentration of free magnesium in the PCR
regards
Magnesium ion act as a cofactor. It cause to specificity and result in better function of polymerase so its role is so important. Final concentration of it is 1.5 mM in the reaction mixture.
Magnesium ion acts as a co-factor for the polymerase, thus enhancing the extension process. It helps in the binding of dNTP to the DNA strand through polymerase.
It is just act as a co-factor for DNA polymerase , with out Mgcl2 we are facing a trouble in annealing .
It helps to keep the enzymes such as Taq Polymerase in their optimal confirmation
Dear,
two things....
1) The polymerase requires a divalent cation (in this case Mg++ from the MgCl2) in order to function.
2) Its concentration must be optimized for every primer:template system. Many components of the reaction bind magnesium ion, including primers, template, PCR products and dNTPs. The main 1:1 binding agent for magnesium ion is the high concentration of dNTPs in the reaction. Because it is necessary for free magnesium ion to serve as an enzyme cofactor in PCR, the total magnesium ion concentration must exceed the total dNTP concentration. Typically, to start the optimization process, 1.5 mM magnesium chloride is added to PCR in the presence of 0.8 mM total dNTPs. This leaves about 0.7 mM free magnesium for the DNA polymerase. In general, magnesium ion should be varied in a concentration series from 1.5-4.0 mM in 0.5 mM steps
Thank you all for the answer and I really appreciate the help. It was really helpful.
Hello Aldosky,
MgCl2 is a cofactor whose Mg+2 interacts with the Taq polmerase and hence enhances its acivity. You can use the conc. between 2-4 mM .
regards
Jitendra
Magnesium is required as a co-factor for Taq polymerase. Because it’s a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction. Optimum mg concentration is very crucial because some of the components of the reaction mixture such as template concentration, dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the amount of free magnesium present thus reducing the activity of the enzyme. Excessive magnesium concentrations stabilize the incorrect bound Primers and so results in decreased specificity of the reaction. Excessive magnesium concentrations also prevent complete denaturation of the DNA during PCR because of its tendency to stabilize double stranded DNA and thus reduce the product yield.
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