Mehdad

It is well appreciated that Mg2+ stabilizes duplex DNA 80 to 100 fold to as much as 140-fold (1). How ever Promega (2) and other online calculators of primer Tm do not take into account the effects of magnesium on helix stability. I personally ran a multiplex PCR which its annealing temperature is close to Tm calculated without magnesium effect. I used Wetmore and Sninsky (1995) N-N formula with a little change in [salt] calculation, [salt]= [K+] + [NH4]. Now my question is, do we need to consider magnesium effect for Tm calculation of primers in PCR? since doing so raises Tm about 5-10 oC? [PLEASE SUPPORT YOUR ANSWERS WITH PUBLISHED ARTICLES IF POSSIBLE] References (1) Nakano S, Fujimoto M, Hara H, Sugimoto N. Nucleic acid duplex stability: influence of base composition on cation effects. Nucleic Acids Res 1999;27:2957–65. (2) http://www.promega.com/biomath/calc11.htm#salt_Desc (3) Wetmur, J.G., and J.J. Sninsky (1995). Nucleic acid hybridization and unconven- tional bases. In PCR Strategies (M.A. Innis, D.H. Gelfand, and J.J. Sninsky, eds.). Acade mic Press, San Diego, CA, pp. 69–83.

Thanks

ummm thanks, could you support your explanation with some published articles if there's any? ummm thanks, could you support your explanation with some published articles if there's any?

What is the effect of multiple freeze-thaw cycles on genomic DNA?

Hi, name's Mehdad, I'm a human and so happy to be ooh and btw I don't like the way we are living.