I'm using a new High-Resolution Melting technique to rapidly identify polymorphisms with a single, unlabeled probe and LCGreen Dye. The technique requires that the two primers and probe must be diluted to 1uM and 5uM, and then only 1ul is placed into the reaction for a final concentration of 0.1uM and 0.5uM. What I have discovered is that if I make a dilution of primer/probe mix, letting it sit for two days and using it creates some non-specific amplification, and letting it sit for a week or more yields no product at all, despite it creating lots of product when it was first created.... Is it possible that the oligonucleotides are degrading because they're in such a low concentration? If so, can I do anything other than make a fresh dilution every time I want to do an assay?
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1 reply to this topic
Posted 13 July 2010 - 08:06 AM
Prepare your primers in TE (Tris-HCl 10 mM, pH 7.5, EDTA 1 mM), or perhaps better, TE 10:0.1 (Tris-HCl 10 mM, pH 7.5, EDTA 0.1 mM). The Tris keeps the water slightly basic and the EDTA removes Mg++ ions necessary for DNAses and most other DNA degrading mechanisms to work. These compounds will have little or no effect on your PCR reaction at the level you are adding them, and will dramatically improve your DNA storage lifetime. You can store DNA at room temperature in TE for months or years.