I am trying to set up a LAMP assay (Loop-mediated isothermal amplification) for the detection of E.coli.
I was told that EDTA can chelate the Mg in my reaction and thus prevent the assay from working (similarly to PCR). Therefore, I hesitate to use TE buffer. Can I prepare my primer mix and extract DNA (using the boiling method) in DNAase free water?
will that affect the quality of my DNA and primers during storage?
Thanks a lot!
Alex
Although this is theoretical a problem, in fact it isn't one. Lets do the calculation around it. EDTA complexes equimolar amounts of bivalent cations (e.g. Magnesium), so 1mM EDTA will complex 1mM Magnesium ions. Lets assume that the PCR reaction contains 2mM Magnesium.
A typical PCR reaction I do has a total volume of 25ul and contains 1ul DNA template in TE buffer (10mM Tris, 1mM EDTA). The final reaction contains 1/25th mM (or 0,04mM or 40nM) EDTA which can complex 0,04mM Magnesium. So instead of running the reaction with 2mM Magnesium, you do it with 1,96mM, which will not be noticable.
Additionally other errors in setting up (pipettes not accurately calibrated, not setting small amounts into the liquid etc.) easily add up to 5-10% error in total, which has a much higher impact in the Magnesium concentration of the reaction than the EDTA. If you use much higher template concentrations (which is usually not necessary), then you can correct for this, but normally this is not necessary.
The conventional Eiken LAMP formulation has an excess of Mg2+. 5 µl of TE with EDTA at 1 mM going into a 25 µl reaction will have minimal impact. In any case the LAMP formulation can be up adjusted in Mg2+ to compensate, but I doubt this is even necessary. Besides, bugs will lyse better in the boiling method with EDTA present and it will hinder nucleases.
