Preparation of template DNA

Since cycle sequencing can be performed using very little template DNA, only very small amounts of detrimental impurities are likely to be carried along with the DNA. Therefore, though still important, template purity may not be as crucial for cycle sequencing as it is for non-cycle sequencing.

Preparation of single-stranded template DNA

Single-stranded template DNA of good purity is essential for excellent sequencing results. Several popular plasmid cloning vectors contain the same lac-derived cloning region as the M13mp vectors and a single-stranded phage replication origin. Production of single-stranded DNA from these vectors is similar to that of the M13 phage and the single-stranded DNA produced can also be used as template for sequencing.

Preparation of double-stranded plasmid DNA

Sequencing double-stranded templates with the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit works effectively with no changes in the reaction protocol. Alkaline denaturation is not required for plasmid DNA templates. For best results, purified plasmid DNA should be used—CsCl gradients, PEG precipitation, adsorption to glass, columns, and other common DNA purification methods all produce suitable DNA. (However, since such small quantities of DNA are added to the reactions, even impure DNA samples can sometimes yield acceptable sequence data.) There are many popular protocols for purifying plasmid DNA from 2-10ml cultures. We have had consistent success with ‘boiling’ (21) and ‘alkaline’ (22) mini-prep methods.

Cycle conditions and template quantity

The temperatures used for cycling the termination reactions should be determined from the characteristics of the sequencing primer, the template, and the length of the termination product desired. The number of cycles required will depend on the quantity and quality of the template DNA used. The following guidelines should assist in choosing cycling parameters.

Cycling temperatures

The melting temperature of the primer should be kept in mind when choosing cycle temperatures. The control primer included in the kit is moderately long (23 bases) with 50% G/C content. The melting temperature of this primer is ~73°C under sequencing reaction conditions, and excellent results are achieved by cycling between 60°C and 95°C. The duration of the steps does not seem to be critical, and even brief pauses (1-10 seconds) at these temperatures seem to be effective (except with dITP as described above).

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