The exact primer concentration and reaction temperatures (up to 50 °C for first strand synthesis and second strand synthesis) strongly depend on the custom primer(s) used. These need to be optimized specifically for each set of custom primers.
NOTE: The higher the primer concentration the higher the likelihood of non-specific priming.
First Strand Synthesis
The final concentration of a target-specific reverse transcription primer should be around 12.5 nM – 1.25 µM final concentration (in this case it would mean you would need in the first strand synthesis reaction.
To achieve this concentration range, you would add e.g., 5 µl of a 50 nM – 5 µM target-specific primer ). to the reaction.
ATTENTION! The total concentration of oligos in the first strand synthesis reaction should not exceed 2 µM. The higher the primer concentration the higher the likelihood of unspecific binding. However, the exact primer concentration and reaction temperature (up to 50 °C for first strand synthesis and second strand synthesis) strongly depends on the custom primer(s) used and has to be optimized accordingly.
Second Strand Synthesis
The final concentration of a target-specific second strand synthesis primer should be 0.5 µM final concentration of the target primer (i.e., 2 µl of 7.5 pM µM Custom Target Primer).
ATTENTION! The total concentration of all oligos in the second strand synthesis reaction should not exceed 2 µM. The higher the primer concentration, the higher the likelihood of unspecific binding. The
The annealing temperature should be chosen according to the Tm of the targeted primers and can range from 45 °C to °- 72 °C. The extension temperature should be 72 °C.