I think there are some points to do in Touch down protocol
Keeping all reactions cold until thermal cycling starts is crucial to avoiding non-specific priming even with TD-PCR.
A hot start setup is preferred. Since the main aim of TD-PCR is to eliminate non-specific interactions during the initial cycles, it is important to use a hot-start set up.
TD-PCR can address problems with monoplex reactions better than multiplex reactions.
Total number of PCR cycles, including the touchdown phase should be kept low (below 35). Too many cycles will lead to appearance of non-specific bands in the gel.
An extra 1 min denaturation cycle at 96°C or 97°C may be extremely useful for difficult templates. BIG REGARDS
I think there are some points to do in Touch down protocol
Keeping all reactions cold until thermal cycling starts is crucial to avoiding non-specific priming even with TD-PCR.
A hot start setup is preferred. Since the main aim of TD-PCR is to eliminate non-specific interactions during the initial cycles, it is important to use a hot-start set up.
TD-PCR can address problems with monoplex reactions better than multiplex reactions.
Total number of PCR cycles, including the touchdown phase should be kept low (below 35). Too many cycles will lead to appearance of non-specific bands in the gel.
An extra 1 min denaturation cycle at 96°C or 97°C may be extremely useful for difficult templates. BIG REGARDS
It is a method for increasing specificity of PCR reactions. Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced (e.g. 1-2°C /every second cycle). The initial annealing temperature should be several degrees above the estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.