Sanger sequencing by capillary electrophoresis

We provide sequencing on the ABI Prism 3100xl genetic analyzer. This is is a 16-capillary instrument using fluorescence based electrophoresis technology for DNA sequencing and fragment analysis. For the cycle sequencing we routinely use the 3'-BigDye dideoxynucleotide triphosphates labeling chemistry.

The robust Sanger sequencing chemistry, proven through decades of results, employs fluorescence-based cycle sequencing requires a DNA template, a sequencing primer, a thermal stable DNA polymerase, nucleotides (dNTPs), fluorescently labeled 3'-BigDye dideoxynucleotides (ddNTPs), and buffer. The components are combined in a reaction that is subjected to cycles of annealing, extension, and denaturation in a thermal cycler. Thermal cycling the sequencing reactions creates and amplifies extension products that are terminated by one of the four dideoxynucleotides. The ratio of deoxynucleotides to dideoxynucleotides is optimized to produce a balanced population of long and short extension products.

To submit samples for sequencing

  • Reffer to the 'Instructions for capillary sequencing samples submission' for the amounts and concentrations of template to be submitted for sequencing and tips for template preparation and primer design.
  • Download the 'Capillary Sequencing Sample Submission Form' (Excel format), fill it in completely, and submit it to the MCIC electronically.
  • Limit sample name to 5 characters and do not use spaces, comas, period or other punctuation in the sample name, or do not start the name with the letter "M", because the sequencing software does not accept them.
  • Bring your samples to the facility, Selby Hall room #009 and place the samples in the refrigerator with the sign 'Sequencing samples'.