Thermocycle sequencing uses Perkin-Elmer/Applied Biosystem's AmpliTaq-FS DNA polymerase and Big Dye terminators with dITP. For problem templates, alternative sequencing chemistries (Big Dye terminators with dGTP, dRhodamine terminators) are available, but a request for alternative sequencing strategies must be discussed in person with staff at the DNA Sequencing Facility prior to submitting a request form. Specifically, Big Dye terminators with dITP may have problems in sequencing G-T repeats and homopolymeric stretches of A or T.

Big Dye terminators can be used in combination with any primer, although careful selection of primers can make the difference between good base calling and a failed sequence. You must supply that primer (10ul of a 0.8 pmole/ul solution for each sample). An 18-mer or greater is recommended. Perkin-Elmer/ABI makes the following recommendations concerning primer synthesis on their own synthesizers: 1) Polystyrene 40-nmole columns are the best choice. 2) If the stepwise efficiency of synthesis is greater than 98%, then primers can probably be used without purification. Otherwise, primers should be purified, e.g. using OPC columns.