magnesium concentration in pcr - (Oct/01/2007 )
So, one of the things that can be changes in order to optimize the pcr is magnesium concentration.
A basic question from a beginner: WHY??
Isn't magnesium for the enzym to work optimal. Isn't that optimal the same whatever primers and templates used?
Mg2+ is essencial for PCR however, usually you have to optimize the concentration. Increasing Mg2+ conc. will decrease stringency, however this ion is essencial for enzyme to bind template. Optimal concentration is when you have specific product, without unspecific bands.
To put it simply it has all to do with the template. Sometimes the template it is more diluted (requiring more Mg ion), sometimes it is more contaminated with PCR inhibitory molecules (also more Mg ions to compensate), and sometimes you want alot of product and you increase the dNTP concentration, which requires the Mg concentration to similarly increase(dNTP chelate Mg ions).
Mg ion tend to make the polymerase more active, but higher activity means more non specific products are made. As your quality of DNA changes so to does the amount of Mg ions you need to add to get a strong specific product band.
The optimum magnesium concentration for yield of PCR product can vary depending on the template purity and concentration, whether or not the primers are HPLC-purified, whether or not you are using SYBR Green I, the dNTP concentration, the composition of the reaction buffer, and the enzyme concentration. Primer annealing temperature increases with increased magnesium.
Fidelity is also affected by the magnesium concentration. Minimal magnesium and dNTPs encourage higher fidelity when the enzyme is barely processive, while normal to high magnesium and dNTPs may lead to lower fidelity.
Too much magnesium can lead to more primer diimer.
Too much magnesium will stabilize double stranded DNA and may prevent complete denaturation of the DNA.
Too much magnesium can also lead to smearing of the PCR product on gels.
Thanks for your good answers!