Primers should be at least 18mer and desalted. Each sample will require 10 picomoles primer per reaction. Calculate the melting temperature of your primer. One of several methods is:
Tm = [(A+T) x 2°C] + [(G+C) x 4°C]
This will determine the cycle sequencing program used to incorporate the fluorescent dideoxynucleotides.
10 pmole/rx (1ul of 10uM) sequencing primer is used.
Optimal results are obtained when primers are diluted in dH2O 10mM Tris buffer (Avoid TE buffer)