To separate the homoduplexes and heteroduplexes, DGGE fragments are run on acrylamide gels. Gels with 9% acrylamide ensure sharp bands and are easy to handle. To pour these gradient gels, two types of stock solution are used: 9% 37.5:1 acrylamide/bisacrylamide in 0.5X TAE and the same stock solution with 7M urea and 40% formamide. The former is called 0% denaturant and the latter is called 100% denaturant.
Gradients from 100% to 0% are rarely used because, in such a broad gradient, the denaturing points of the homoduplexes and heteroduplexes would probably be very close to each other; therefore, a range of 30% for the gradients is recommended. To select a urea/formamide gradient, the predicted melting temperature (Tm) of a product as obtained from the computer analysis is used in the empirical formula Tm × 3.2−182.4 = % denaturant.
The melting point is positioned approximately in the center of the gel by simply adding and subtracting 15% from this calculated urea/formamide concentration to obtain the desired 30% gradient. Acrylamide gels with gradients of urea and formamide are poured using a simple gradient mixer that consists of two reservoirs that are connected at the base with a short tube. Such a system is widely used for pouring all types of gradient.