The formation of this kind of structures requires a high-resolution master, typically generated by conventional nanofabrication techniques that can be replicated by molding or embossing [35].
Briefly, 20 ng of genomic DNA (10 ng μl 1) were amplified in total volume of 20 μl with 10 μl high-resolution master mix, 2.4 m M MgCl2, and 0.25 m M each of oligonucleotide primers, 2 μl template DNA and 5.2 μl dH2O.
Briefly, 20 ng of genomic DNA (10 ng μl 1) were amplified in total volume of 20 μl with 10 μl High-Resolution Master Mix, 2.4 mM MgCl2, and 0.25 mM each of oligonucleotide primers, 2 μl template DNA and 5.2 μl dH2O.
Reaction mixtures contained 10 ng genomic DNA, 0.25µM of each primer, 3mM MgCl2 and 1× LightCycler 480 High Resolution Melting Master Mix (Roche Diagnostics, Meylan, France) containing Taq polymerase, nucleotides and the dye Resolight in a total volume of 10µl.
The reaction was performed in a 15 µl PCR mix containing 1X LightCycler® 480 High Resolution Melting Master Kit (Roche), 3.5 mM MgCl2, 0.5 µM of each primer and between 10 and 20 ng of DNA The amplification protocol consisted of a first denaturation step at 95°C [5 min], 45 cycles of denaturation at 95°C [10 s], annealing at 55°C [30 s], and extension at 72°C [30 s].
Amplification was performed on the ViiA7™ Real-Time PCR System Life Technologiess) using the LightCycler® 480 High Resolution Melting Master Mix (Roche Diagnostics).
DNA was amplified in a total volume of 10 μl, including 1 μl (5 30 ng) of genomic DNA, 0.2 µM of each primer, 4.2 mM MgCl 2, and 1× Roche High Resolution Melting Master kit mix.
HRM analysis was performed on a LightCycler 480s (Roche Diagnostics, Penzberg, Germany) in a reaction mixture that contained 10 ng wild-type DNA, 10 ng mutant DNA, 0.5 mM of each primer, and 3 mM MgCl2 in the LightCycler 480 High Resolution Melting Master containing ResoLight dye (Roche Diagnostics) adjusted to a total volume of 10 μl with PCR-grade water.
Amplification for HRMA was performed on 2 μl of 1 1000 dilutions of previously amplified clones using the same primers as in the first PCR reaction in a 10 μl reaction volume containing 1× LightCycler® High Resolution Melting Master (Roche), 2 mM MgCl2, and 3 pmol of each primer.
Nested PCR were performed by using Light Cycler 480 (Roche Applied Science) in a total volume of 10 μl of the mixture containing 1 μl of diluted LR product, 1 × Light Cycle 480 High resolution melting master mix (Roche Applied Science) and 5 pmol of each primer.
