As another example, when using the universal -40 17-mer, which has a melting temperature of about 50°C, cycling between 45°C and 95°C is effective. If in doubt, choose a wide temperature range with pauses (15-30 seconds) at the extremes of temperature.
The termination reaction cycles should always have a denaturation temperature of 95-98°C (however, avoid extended steps at 98°C since at this temperature the enzyme has a half-life of less than one hour). Since the optimum temperature for polymerization is about 70-75°C, 72°C is a good choice for the termination step (except when using dITP, which requires a maximum temperature of 55-60°C). An annealing step (e.g. <60°C) is required only with primers less than ~24 bases.
Number of cycles and quantity of template
The number of cycles required will primarily depend on the amount of template DNA (in fmols) used for sequencing. It will also depend on the purity of the DNA, and the sensitivity of autoradiographic detection. The minimum quantities of highly-purified DNA which we have been able to sequence using these methods are about 5fmol of M13mp18 DNA and about 15fmol of pUC18 DNA. (For routine sequencing, we recommend 25fmol of M13 and 75fmol of plasmid DNA). When sequencing very small amounts of template, it has been observed that the number of cycles has a strong influence on sequence intensity. Increasing the number of cycles from 30 to 60 will increase the signal significantly when using less than ~50fmol of template DNA, whereas increasing the number of cycles with more than ~100fmol is of little benefit, and may even produce background sequence. So in general, use more cycles when template amounts are limited. Also, a modest improvement can sometimes be achieved by increasing the amount of primer 2-5 fold. It is undesirable to use too much template as the result will be a shortened sequence extension. Figure 1 shows the result of increasing template quantities to an excess.
Designing a new sequencing primer
The length of the primer (and its sequence) will determine the melting temperature and specificity. For the cycling temperatures normally used, the primer should be about 18-25 nucleotides long. It is also a good idea to check the sequence of the primer for possible self-annealing (dimer formation could result) and for potential ‘hairpin’ formation, especially those involving the 3' end of the primer. Finally, check for possible sites of false priming in the vector or other known sequence if possible, again stressing matches which include the 3' end of the primer.