Doing the standardization of the amplification of the transcript of interest, I observed that the more I reduced magnesium concentration, the more amplification I got of my transcript. So I decided to evaluate a condition without magnesium and as you can see at the image, the amplification was greater than with 0.5 mM, with higher annealing temperatures.
Magnesium is required by DNA and RNA polymerases as a cofactor. If you're seeing amplification without directly adding magnesium to the reaction, it's probably coming from somewhere else - magnesium is included in most PCR buffers, and there is usually also a small amount in the storage buffer for the polymerase. Since your reaction seems to work better without supplemental magnesium, I don't see any reason to add it :)
Magnesium is required by DNA and RNA polymerases as a cofactor. If you're seeing amplification without directly adding magnesium to the reaction, it's probably coming from somewhere else - magnesium is included in most PCR buffers, and there is usually also a small amount in the storage buffer for the polymerase. Since your reaction seems to work better without supplemental magnesium, I don't see any reason to add it :)
It is also possible that the enzyme that you are using can utilise other divalent cations like Mn which may be present in one of the other reagents ( buffer or the dna perhaps) but the most likely reason is as Laura Leighton says
- Agree with both previous answers especially Laura
- It is a co factor so you require magnesium f or the enzymes to operate
- Thats said as long as your magnesium is 2-3mM that mg can be derived from any where
- The more Magnesium the more specific product you make; but also more non specific products
- This is why 2-3mM is considered optimal
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