The BigDye® Terminator v3.1 Cycle Sequencing Kit provides all the required reagents for a sequencing reaction in a pre-mixed format. All you provide is your template and template-specific primer. The reagents are suitable for performing fluorescence based cycle sequencing reactions on single-stranded or double-stranded DNA templates, PCR fragments, and large templates.
This fluorescence-based method is a variation of the Sanger chain-termination protocol developed over 20 years ago. It employs four different dye-labeled dideoxynucleotides (ddNTPs) that correspond to each of the four DNA nucleotides (dNTPs). During amplification, when a dNTP is incorporated, the new fragment will continue to grow. A ddNTP however, contains a hydrogen atom instead of a hydroxyl group at its 3’ end, preventing the phosphodiester bond from forming between it and the next incoming nucleotide. Therefore, when a ddNTP is incorporated, further chain elongation is blocked and this results in a population of truncated products of varying lengths. The reaction mixture contains a mixture of dNTPs and ddNTPs at concentrations that create a statistical probability that a ddNTP will be incorporated instead of a dNTP at each nucleotide position in the newly generated fragments.
One reaction therefore contains thousands of fragments of lengths differing by one nucleotide. Once loaded onto the instrument, these fragments migrate according to size on a 4% acrylamide gel, eventually crossing paths with a laser. The laser excites the fluorescent labels, and the result is a contiguous read of labeled bases that represent the sequence of the template DNA.
The pieces I am sequencing are very small DNA aptamers, about 60 bp. I first amplify them by standard PCR and then ligate them into a thymidine-overhang vector. (no purification step is necessary prior to ligation.) Transformations are streaked to LB Amp plates. The following day, cells containing the desired plasmid are harvested and DNA is isolated by miniprep and ultimately recovered from the miniprep supernatant by isopropanol (IPA) precipitation. The DNA is then crudely checked for concentration and purity using agarose gel electrophoresis against known standards. If clean bands are observed, 0.5 ul neat template (from the IPA precipitation, dissolved in 10 ul TE buffer) is used in each reaction. Our primers are 15 bases long, ordered from Sigma Genosys. They arrive ready to use, no purification is necessary. I follow the sequencing protocol provided by Applied Biosystems and always obtain good results; I have yet to encounter any problems with this kit. I never use a control, and I would highly recommend this kit to all of my colleagues.