A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes.
This consists of a pre-densification step using a thermal-gradient chemical vapor infiltration process followed by a high-pressure pitch impregnation and carbonization process.
This consists of a pre-densification step using a thermal-gradient chemical vapor infiltration process followed by pitch impregnation/carbonization cycles carried out under high pressure.
DNA was amplified by using a thermal cycler (Touchgene Gradient, Techne Inc., Burlington, NJ, USA).
Using a thermal cycler (Master cycler ep gradient, Eppendorf) PCR was performed using 1 cycle of 95°C for 3 min, 40 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 2 min followed by 1 cycle of 72°C for 10 min.
The PCR was conducted using a thermal cycler (Eppendorf Master Cycler Gradient) as follows: heat denaturation at 94°C for 2 min, followed by 45 cycles of PCR (94°C for 30 s, annealing temperature of 60.6°C for 30 s, extension temperature of 72°C for 1.3 min).
Specifically, nested PCR was performed using a thermal cycler (PCR Thermal Cycler Dice Gradient, Takara Bio, Inc., Shiga, Japan) with primers (Sigma-Aldrich, Japan), Taq polymerase (Takara Ex Taq Version; Takara Bio, Inc ., and extracted DNA.
Prior to dHPLC analysis these products were denaturated at 95°C for 5 min and subsequently cooled down using a temperature's gradient until 50°C using a thermal cycler.
We created a thermotaxis setup as described previously using a steep thermal gradient on a thin agar plate [20].
Direct temperature preference and tolerance experiments using a high-pressure aquarium with a thermal gradient have not yet been carried out on adult A. pompejana.
