When do I need to add some extra amount of MgCI2?

Conventional PCR Master Mixes typically have an adequate amount of MgCl2 for standard applications. However, there are situations where a higher concentration may be necessary. This is particularly true when dealing with PCR inhibitors present in DNA extracts that have an affinity for Mg2+ ions. Thus, increasing the MgCl2 concentration helps counteract the effects of these inhibitors, ensuring successful amplification. Additionally, templates with higher GC content pose challenges for amplification under normal conditions. Adding extra amounts of MgCl2 can enhance success in amplifying these templates. For high GC templates, MgCl2 plays a crucial role in reducing the annealing temperature, ensuring accurate primer binding, and preventing rapid renaturation of DNA. Similarly, longer templates (such as those spanning 2Kb or 5Kb) can be difficult to amplify completely. Once again, the addition of extra MgCl2 is useful for facilitating successful amplification of these templates.